A method for removing antibiotics by using a quorum-sensing signal molecule to strengthen an algae-bacteria combination

Abstract

The application provides a method for strengthening removal of antibiotics by using a quorum sensing signal molecule and alga-bacteria combination, and belongs to the fields of environmental biological technology and wastewater treatment technology.The method is used to solve the technical problems of low antibiotic degradation efficiency caused by insufficient electron transfer efficiency and lack of effective means for directional regulation of the electron transfer path in the process of removing antibiotics by using the alga-bacteria combination.The method comprises the following steps: jointly culturing microalgae, activated sludge, wastewater containing antibiotics and N-dodecanoyl homoserine lactone, and reconstructing an electron transfer path to remove the antibiotics.

Description

Technical Field

[0001] This invention belongs to the fields of environmental biotechnology and wastewater treatment technology, and in particular relates to a method for enhancing the removal of antibiotics by algae-bacteria complexes using quorum sensing signal molecules. Background Technology

[0002] Sulfonamide antibiotics (such as sulfathiazole) are among the most frequently detected and highest-concentrated antibiotics in aquatic environments, typically ranging from ng / L to µg / L in municipal wastewater and surface water. These antibiotics exert their antibacterial effect by competitively inhibiting bacterial dihydrofolate synthase. Even trace amounts can exert continuous selective pressure on the microbial community, thereby inducing and spreading antibiotic resistance genes. Traditional biological treatment processes, such as activated sludge processes, are not entirely effective in removing sulfonamide antibiotics. Not only does the removal rate of their parent compounds fluctuate significantly, but some transformation products retain antibacterial activity and may even exhibit stronger ecotoxicity. The potential eco-health risks posed by this incomplete removal have made antibiotic wastewater treatment one of the most pressing problems to be solved in the field of water pollution control. Therefore, there is an urgent need to develop enhanced treatment technologies that can improve the intrinsic degradation capacity of the microbial community.

[0003] In recent years, algae-bacteria consortia have attracted widespread attention as a green and sustainable wastewater treatment platform. In this system, microalgae release O2 through photosynthesis and provide organic carbon sources (extracellular secretions) for bacteria, while bacteria provide CO2 and inorganic nutrients (such as NH4) to the microalgae through mineralization. + -N), forming a close synergistic metabolic relationship between the two (Journal of Hazardous Materials, 2022, 440, 129785). This synergistic relationship can theoretically significantly enhance the metabolic transformation capacity of the consortium for organic pollutants (including antibiotics). However, existing research shows that the degradation performance of this consortium for antibiotics is subject to two key factors: (1) the community structure and environmental adaptability of the consortium, and (2) the efficiency of extracellular electron transfer in microorganisms. Among them, electron transfer is the thermodynamic engine of the oxidative degradation process, that is, the oxidative cleavage of pollutants (especially electron-deficient compounds such as sulfonamides) essentially depends on the ability of microorganisms to transfer reducing equivalents to extracellular receptors (Chemical Engineering Journal, 2025, 165741). Current technology mainly relies on the natural metabolic activity of the consortium and lacks effective means to directionally regulate the rate and pathway of electron transfer, resulting in unstable degradation efficiency and difficulty in meeting the needs of actual wastewater treatment.

[0004] In microbial communities, extracellular electron transfer is primarily achieved through two distinct pathways. The first is a mediator-dependent pathway: microorganisms secrete soluble redox mediators (such as riboflavin, phenazine, and quinone compounds) into the extracellular space. These mediators diffuse back and forth between the cell and electron acceptor, shuttle-transferring electrons. This model relies on the synthesis-secretion-diffusion-turnover process of the mediator and is susceptible to diffusion limitations and mediator consumption. The second is a solid-state direct pathway: microorganisms directly transfer electrons to acceptors through physical contact, via outer membrane cytochrome proteins (such as MtrC / OmcA) or conductive extracellular polymers. This model does not rely on soluble mediator diffusion, resulting in a higher and more stable electron transfer rate and promoting the formation of tight intertrophic aggregates. Current technologies generally lack understanding and regulatory mechanisms for the switching between these two pathways. In algal-bacterial complexes, the regulatory logic of how the reducing equivalents produced by microalgal photosynthesis flow to bacteria through these pathways and drive pollutant degradation remains unclear. If a conditional signal can be found to actively switch the electron transport pathway of the consortium from a slow, easily damaged, mediator-dependent type to a fast, stable, solid-state direct type, it is expected to significantly improve the degradation efficiency of antibiotics.

[0005] Quorum sensing is a density-dependent communication system widely present in Gram-negative bacteria, where bacteria coordinate gregarious behavior by secreting and sensing N-acylhomoserine (AHL) lactone signaling molecules (Bioresource Technology, 2022, 127113). In recent years, preliminary evidence has emerged that exogenous addition of AHLs can affect biofilm formation and the expression of specific degradative enzymes (Environmental Science & Technology, 2022, 12645-12655). However, there have been no previous reports on whether and how AHLs can systematically reconstruct the electron transport network of algal-bacterial complexes, shifting the electron transport pathway from a mediator shuttle mode to an EPS-conductive network mode. This gap in regulatory mechanisms not only limits the effectiveness of algal-bacterial complexes in antibiotic wastewater treatment but also hinders the engineering application of the technology of signal molecule regulation-electron transport reconstruction-enhanced pollutant degradation.

[0006] To address the shortcomings of the existing technologies, this invention proposes and verifies for the first time: using exogenous C12-HSL as a regulatory switch to upregulate the EPS synthesis gene ( bcsA , pgaA ) and direct electron transport genes ( mtrCThis invention simultaneously inhibits flavin mediator turnover, reconstructing the electron transport pathway of the algae-bacteria consortium from a mediator-dependent model to an EPS-embedded conductive network model, thereby systematically enhancing the consortium's degradation capacity for sulfonamide antibiotics. This invention provides a novel technical approach and experimental basis for actively regulating microbial electron transport to enhance the removal of recalcitrant pollutants.

[0007] In summary, a method for enhancing antibiotic removal by algae-bacteria consortia using quorum sensing signaling molecules is proposed. Summary of the Invention

[0008] In view of this, the present invention aims to propose a method for enhancing the removal of antibiotics by algae-bacteria complexes using quorum sensing signal molecules, in order to solve the technical problems of insufficient electron transfer efficiency and lack of effective means for directional regulation of electron transfer pathways in the existing process of antibiotic removal by algae-bacteria complexes, which leads to low antibiotic degradation efficiency.

[0009] To achieve the above objectives, the present invention employs the following technical solution to provide a method for enhancing the removal of antibiotics by algae-bacteria consortia using quorum sensing signaling molecules, comprising: Microalgae, activated sludge, antibiotic-containing wastewater, and N-dodecyl homoserine lactone were co-cultured to reconstruct the electron transport pathway for antibiotic removal.

[0010] Furthermore, the concentration of the N-dodecyl homoserine lactone is 100-300 μg / L.

[0011] Furthermore, the concentration of the N-dodecanoylhomoserine lactone is 300 μg / L.

[0012] Furthermore, the microalgae mentioned is Chlorella proteoglycans.

[0013] Furthermore, the antibiotic is a sulfonamide antibiotic.

[0014] Furthermore, the activated sludge mainly comprises Rhizobium species. Rhizobiaceae Microbacteriaceae Microbacteriaceae Structuraceae Caulobacteraceae Sphingosomalidaceae Sphingomonadaceae Trichomonaceae Comamonadaceae At least one of them.

[0015] Furthermore, the antibiotic is sulfathiazole.

[0016] Furthermore, the initial dry weight of the microalgae and activated sludge is 500 mg / L.

[0017] Furthermore, the mass ratio of microalgae to activated sludge is 5:1.

[0018] Furthermore, the culture temperature of the consortium is 30±2 ℃, and the light intensity is 200-300 μmol / m². 2 / s, pH 7-8.

[0019] Beneficial effects: 1. By adding C12-HSL to the algae-bacteria consortium, the extracellular electron transport pathway was reconstructed from a mediator-dependent type to an EPS-embedded conductive network type, which significantly improved the degradation efficiency of antibiotics. Experiments showed that at a dosage of 300 μg / L C12-HSL, the 7-day removal rate of sulfathiazole by the consortium increased from 48.5% to 83.1%.

[0020] 2. By reconstructing the electron transport pathway using C12-HSL, the extracellular electron transport capacity of the algal-bacterial complex was significantly enhanced. Conductive atomic force microscopy (C-AFM) results showed that the local current increased from 30.9 pA to 163.1 pA, an increase of 5.3 times.

[0021] 3. While enhancing antibiotic degradation, the photosynthetic activity of microalgae (Fv / Fm>0.60) was not inhibited, ensuring the stable operation of the algae-bacteria complex system and achieving a balance between efficient degradation and system stability.

[0022] 4. Through C12-HSL regulation, key genes for EPS synthesis were upregulated ( bcsA , pgaA ) and direct electron transport genes ( mtrC The expression of ) was reduced, while the accumulation of metabolites related to flavin mediator turnover was downregulated, thus achieving a systematic reconstruction of electron transport pathways at both the gene and metabolite levels. Attached Figure Description

[0023] Figure 1 The comparison chart between Example 1 and Example 2 shows that the key genes for EPS synthesis were upregulated. Figure 2 Comparison chart of Example 1 and Example 2 - downregulation of the accumulation of metabolites related to flavin mediator turnover; Figure 3 A comparison chart of different C12-HSL contents; Figure 4 Comparison chart of Example 1 and Example 2 - electrochemical analysis; Figure 5 This is a comparison of Fv / Fm of algae-bacteria complexes at different concentrations of AHL. Detailed Implementation

[0024] Referring to the accompanying drawings, this embodiment provides a method for enhancing the removal of antibiotics by an algae-bacteria consortium using quorum sensing signal molecules. The method includes: co-culturing microalgae, activated sludge, antibiotic-containing wastewater, and N-dodecyl homoserine lactone to reconstruct electron transport pathways for antibiotic removal.

[0025] The concentration of N-dodecyl homoserine lactone is 100-300 μg / L, preferably 300 μg / L.

[0026] Microalgae are Chlorella pyrenoidosa ( Chlorella pyrenoidosa ).

[0027] Activated sludge originates from the activated sludge in the aeration tanks of wastewater treatment plants, and mainly includes... Rhizobiaceae (Rhizobium family) Microbacteriaceae (Microbacteriaceae) Caulobacteraceae (Styloides family) Sphingomonadaceae (Sphingomonasceae family) Comamonadaceae At least one of the following families: (Trichomonasceae).

[0028] The antibiotic is a sulfonamide antibiotic, preferably sulfathiazole.

[0029] The initial dry weight of microalgae and activated sludge was 500 mg / L, and the mass ratio of microalgae to activated sludge was 5:1.

[0030] The assemblage was cultured at a temperature of 30±2 ℃ and a light intensity of 200-300 μmol / m². 2 / s, pH 7-8.

[0031] Mechanism of action Electron transport pathway reconfiguration: The addition of C12-HSL prompts the extracellular electron transport pathway of the complex to change from a mode dependent on soluble flavin / quinone mediators to a direct electron transport mode dependent on the extracellular polymer (EPS) embedded conductive network.

[0032] Functional gene regulation: Upregulated key genes for EPS synthesis (such as...) bcsA and pgaA The expression of these genes promotes the secretion and structural reorganization of EPS (rich in protein, polysaccharides, and humic acid). It also upregulates direct electron transport genes (such as...). mtrC The abundance of ).

[0033] Metabolite regulation: Downregulated the accumulation of metabolites related to flavin mediator turnover (such as lumichrome).

[0034] Example 1 System construction: Chlorella proteoglycans (FACHB-5) and activated sludge from a municipal wastewater treatment plant were mixed at a mass ratio of 5:1 (algae:sludge), and the initial mixed liquor suspended solids concentration (MLSS) was 500 mg / L.

[0035] Wastewater characteristics: The synthetic wastewater contained an initial concentration of sulfathiazole (STZ) of 10 mg / L, COD of approximately 637 mg / L, and NH4+. + -N approximately 425 mg / L, pH maintained at 7-8.

[0036] Operating conditions: In a column-shaped photobioreactor, the temperature was 30 ± 0.2 ℃, the stirring speed was 200 rpm, 2.5% CO2 was introduced, and the light intensity was 250 μmol / m². 2 / s.

[0037] Signaling molecule addition: C12-HSL was added all at the beginning of the culture to make a final concentration of 300 μg / L in the system.

[0038] Incubation period: 7 days.

[0039] Example 2 Unlike Example 1, C12-HSL is not added.

[0040] Example 3 Unlike Example 1, the concentration of C12-HSL added was 100 µg / L.

[0041] Example 4 Unlike Example 1, the concentration of C12-HSL added was 200 µg / L.

[0042] like Figure 1 As shown, compared with Example 2 (without C12-HSL), the addition of 300 µg / L of C12-HSL resulted in... Figure 1 (a) Genes associated with EET were upregulated; Figure 1 (b) Genes associated with the respiratory chain complex were upregulated; Figure 1 (c) Genes associated with algal-bacterial interactions were upregulated; Figure 1 (d) Genes related to antibiotic degradation and transformation were upregulated.

[0043] like Figure 2 As shown, the differences in electron shuttle metabolites between Example 2 (without C12-HSL) with the addition of 300 µg / L C12-HSL include riboflavin and its derivatives (lucoflavin), phenazine mediators, and quinone / phenolic mediators.

[0044] like Figure 3As shown, compared with Example 2 (without C12-HSL), the addition of 100, 200, and 300 µg / L of C12-HSL improved the antibiotic degradation effect. At the optimal C12-HSL dosage (300 μg / L), the 7-day removal rate of sulfathiazole (STZ) by the combination increased from 48.5% to 83.1%, an increase of 34.6 percentage points.

[0045] like Figure 4 As shown, compared with Example 2 (without C12-HSL), the addition of 300 µg / L of C12-HSL resulted in... Figure 4 (a) Electrochemical analysis showed that C12-HSL altered the electron transport characteristics of microbial-derived EPS. Cyclic voltammetric curves of LB-EPS and TB-EPS differed and varied with culture time and the addition of C12-HSL. Figure 4 (b) Further evidence was provided for the changes in interfacial charge transport induced by C12-HSL. The Nyquist spectra of the LB-EPS and TB-EPS components differed, and the C12-HSL-treated samples generally exhibited lower impedance than the corresponding untreated samples. The decrease in impedance response was consistent with the reduction in charge transport resistance at the EPS-electrode interface. Combined with cyclic voltammetry results, this indicates that C12-HSL restructured the electrochemical environment of the extracellular matrix.

[0046] like Figure 5 As shown, the Fv / Fm ratio of the algae-bacteria complex was compared at the beginning and end of the experiment under different concentrations of AHL. The photosynthetic activity of the microalgae (Fv / Fm>0.60) was not inhibited during the enhanced treatment, ensuring the stability of the system.

[0047] The embodiments of the present invention disclosed above are merely illustrative of the invention. These embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention.

Claims

1. A method for enhancing antibiotic removal by algae-bacteria consortia using quorum sensing signaling molecules, characterized in that, include: Microalgae, activated sludge, antibiotic-containing wastewater, and N-dodecyl homoserine lactone were co-cultured to reconstruct the electron transport pathway for antibiotic removal.

2. The method for enhancing antibiotic removal by algae-bacteria consortia using quorum sensing signaling molecules according to claim 1, characterized in that: The concentration of the N-dodecanoylhomoserine lactone is 100-300 μg / L.

3. The method for enhancing the removal of antibiotics by algae-bacteria consortia using quorum sensing signaling molecules according to claim 2, characterized in that: The concentration of the N-dodecanoylhomoserine lactone was 300 μg / L.

4. The method for enhancing antibiotic removal by algae-bacteria consortia using quorum sensing signaling molecules according to claim 1, characterized in that: The microalgae mentioned is Chlorella proteoglycans.

5. The method for enhancing antibiotic removal by algae-bacteria consortia using quorum sensing signaling molecules according to claim 1, characterized in that: The activated sludge mainly contains Rhizobium family. Rhizobiaceae Microbacteriaceae Microbacteriaceae Structuraceae Caulobacteraceae Sphingosomalidaceae Sphingomonadaceae Trichomonaceae Comamonadaceae At least one of them.

6. The method for enhancing antibiotic removal by algae-bacteria consortia using quorum sensing signaling molecules according to claim 1, characterized in that: The antibiotic in question is a sulfonamide antibiotic.

7. The method for enhancing antibiotic removal by algae-bacteria consortia using quorum sensing signaling molecules according to claim 6, characterized in that: The antibiotic in question is sulfathiazole.

8. The method for enhancing antibiotic removal by algae-bacteria consortia using quorum sensing signaling molecules according to claim 1, characterized in that: The initial dry weight of the microalgae and activated sludge was 500 mg / L.

9. The method for enhancing antibiotic removal by algae-bacteria consortia using quorum sensing signaling molecules according to claim 8, characterized in that: The mass ratio of microalgae to activated sludge is 5:

1.

10. The method for enhancing antibiotic removal by algae-bacteria consortia using quorum sensing signaling molecules according to claim 1, characterized in that: The consortium was cultured at a temperature of 30±2 ℃ and a light intensity of 200-300 μmol / m². 2 / s, pH 7-8.

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