A method and application for enhancing biofilm stability in wastewater treatment systems based on quorum sensing

Abstract

This invention discloses a method and its application for enhancing biofilm stability in wastewater treatment systems based on quorum sensing. The method involves adding the quorum sensing signaling molecule N-acylhomoserine lactone to the biofilm culture system. This method uses exogenously added quorum sensing signaling molecules to enhance the function of the biofilm in secreting extracellular polymers, forming a protective layer on the biofilm surface that can withstand the impact of water quality changes, resulting in a biofilm with excellent stability. Through this method, this invention not only enhances the stability of the biofilm in response to environmental and water quality fluctuations but also optimizes the microbial community structure and metabolic characteristics of the biofilm. The process is convenient and simple, requiring no complex control processes, and can be carried out simultaneously during the biofilm initiation process, overcoming the bottleneck of traditional methods that rely on continuous carbon source addition for biofilm stability control. The biofilm control technology developed in this invention has broad application prospects in the field of wastewater treatment.

Description

Technical Field

[0001] This invention relates to the field of environmental microbiology technology, specifically to a method and application for enhancing biofilm stability in wastewater treatment systems based on quorum sensing. Background Technology

[0002] With the continuous development of society, water pollution has become a global problem, and large amounts of high-salinity wastewater are generated from daily life, food processing, metallurgy, and chemical industries. Microorganisms are a diverse group of life forms that are widely distributed in nature and possess a variety of specific functions. For example, electroactive microorganisms can achieve energy / matter transfer through extracellular bidirectional electron transfer at a relatively low energy consumption level, thus meeting the contemporary demand for improving the quality and efficiency of wastewater treatment. They have enormous development potential and provide new research ideas for the application and expansion of novel bioremediation technologies for water pollution.

[0003] Microbial methods are currently the most important and widely used approach in water pollution control and water purification processes, with the physiological and biochemical reactions and behavioral regulation of microorganisms directly affecting their wastewater treatment efficiency. However, biofilm structures are highly susceptible to water quality fluctuations, significantly impacting microbial activity and thus reducing the biochemical treatment efficiency of the biofilm. Quorum sensing (QS) is an interspecies communication process guided by specific chemical signals (i.e., quorum sensing signal molecules), which can actively alter the structure and physicochemical properties of microbial communities, thereby enhancing biofilm performance. Currently, effective methods for strengthening biofilm protection mechanisms against water quality fluctuations based on quorum sensing still lack effective approaches.

[0004] Therefore, it is extremely important to develop a feasible, economical, effective, and targeted biomembrane stability regulation technology. Summary of the Invention

[0005] In view of the shortcomings of current water treatment processes, such as long start-up time and poor stability of biofilms under natural conditions, the purpose of this invention is to provide a method and application for strengthening the stable regulation of environmental microorganisms with biofilm as the core, and to provide an effective technical means to enhance microbial activity and improve the efficiency of biological wastewater treatment.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] The first objective of this invention is to provide the application of the quorum sensing signaling molecule N-acylhomoserine lactone in improving the salt shock resistance of microbial biofilms, wherein the N-acylhomoserine lactone is N-(3-oxododecyl)-l-homoserine lactone.

[0008] The second objective of this invention is to provide a method for enhancing biofilm stability in a wastewater treatment system based on quorum sensing, comprising the following steps: adding the quorum sensing signal molecule N-acylhomoserine lactone to a biofilm culture system, and culturing the biofilm, wherein the N-acylhomoserine lactone is N-(3-oxododecyl)-l-homoserine lactone.

[0009] Preferably, the concentration of the N-acylhomoserine lactone is 5–15 μmol / L.

[0010] Preferably, the biofilm culture system consists of a nutrient culture medium, carbon-based materials, and activated sludge. The nutrient culture medium formula is 0.31 g / L NH4Cl, 0.13 g / L KCl, 2.27 g / L NaH2PO4·2H2O, mineral solution, vitamins, and 10 mmol / L sodium acetate, with deionized water as the solvent.

[0011] Preferably, the quorum sensing signal molecule serves as a regulatory factor to enhance microbial stability, and the biofilm is a biofilm system formed from the initiation stage to domestication maturity.

[0012] A third object of the present invention is to provide a biofilm obtained using any of the methods described above.

[0013] A fourth objective of this invention is to provide the application of the above-described biofilm in the treatment of sewage or wastewater.

[0014] Preferably, the wastewater or sewage has a salt concentration of 3% to 10% by mass, and the effective period of the salt resistance of the biofilm is 1 to 2 months.

[0015] Preferably, the salt is one or more selected from NaCl, CaCl2, Na2SO4, and MgSO4.

[0016] Preferably, the treatment of wastewater or sewage includes the removal of organic pollutants, with a chemical oxygen demand (COD) load of 50–1000 mg / L. The wastewater originates from domestic water use, and the sewage originates from tailwater from food processing, metallurgy, or chemical industries.

[0017] Compared with existing biofilm stability regulation technologies, this invention employs a method that is simple, feasible, economical, and efficient. It enhances biofilm stability by adding an exogenous quorum sensing signal molecule, acylhomoserine lactone, to the microbial growth system. Laser confocal electron microscopy scanning of mature biofilms revealed that the quorum sensing signal molecule induces a more uniform and dense biofilm with a higher proportion of highly active microorganisms. Furthermore, treatment of wastewater containing 10% salt concentration with this biofilm did not reduce microbial activity, demonstrating that the quorum sensing signal molecule has an indirect protective effect on the biofilm and significantly improves its high-salt adaptability. This invention achieves environmental microbial stability regulation centered on the biofilm, improves the efficiency of microbial wastewater treatment, and, most importantly, reveals the self-protection mechanism of the biofilm under external water quality shocks. It establishes an economical, feasible, and effective method for optimizing and regulating biofilms, showing promising application prospects.

[0018] This invention employs exogenously added quorum sensing signaling molecules to enhance the function of biofilm secretion of extracellular polymers, enabling the formation of a protective layer on the biofilm surface. This layer helps the biofilm cope with the impact of water quality changes, resulting in a biofilm with excellent stability. Through this method, this invention not only enhances the stability of biofilms in response to environmental and water quality fluctuations but also optimizes the microbial community structure and metabolic characteristics of the biofilm. The process is convenient and simple, requiring no complex control techniques, and can be carried out simultaneously during the biofilm initiation process. It overcomes the bottleneck of traditional methods that rely on continuous carbon source addition for biofilm stability control, providing a highly efficient, stable, inexpensive, and ecologically safe method for biofilm stability control. The biofilm control technology developed in this invention has broad application prospects in the field of wastewater treatment. Attached Figure Description

[0019] Figure 1 This is a laser confocal imaging (CLSM) of biofilms formed under different culture conditions in Example 1 of the present invention, where red and green pixels represent dead cells and live cells, respectively.

[0020] Figure 2 This illustrates the effect of QS signal molecular regulation on COD removal from catering wastewater in Example 2 of the present invention.

[0021] Figure 3 This is a schematic diagram of the microbial electrochemical system reaction device in Embodiment 3 of the present invention.

[0022] Figure 4 This is a schematic diagram showing the change of current density over time during biofilm formation under different culture conditions in Example 3 of the present invention.

[0023] Figure 5 This is the first current cycle of the biofilm in Example 3 of the present invention after a short-term high salinity shock (10% salinity by mass, 4 hours).

[0024] Figure 6 This is a schematic diagram showing the change of current density over time in a mature biofilm in saline wastewater (10% salinity by mass) in Example 3 of the present invention.

[0025] Figure 7 This illustrates the effect of QS signal molecular regulation on COD removal from municipal wastewater in Example 4 of the present invention. Detailed Implementation

[0026] The present invention will be further described below with reference to embodiments. These embodiments are intended to illustrate the invention and are not intended to limit the invention in any way.

[0027] Example 1:

[0028] QS signaling molecules promote the domestication and initiation of biological membranes:

[0029] The biofilm culture system consisted of 100 mL of nutrient medium, carbon-based material (graphite plates, 2.5 cm × 3 cm × 0.1 cm, Beijing Jinglong Special Carbon Technology Co., Ltd., were used in this embodiment), and natural environmental sediment sludge (inoculation amount of 50 mg / L in each reactor, sediment sludge was dredged from 10–20 cm below the water surface of a long-running sediment microbial fuel cell in a wastewater treatment plant). The culture was conducted under anaerobic conditions in 100 mL blue-mouthed bottles with sealed membranes. The natural environmental sediment sludge served as the microbial inoculum, and the biofilm formed on the carbon-based material carrier. The nutrient medium was artificially prepared with the following formula: 0.31 g / L NH4Cl, 0.13 g / L KCl, 2.27 g / L NaH2PO4·2H2O, mineral solution (Table 1), vitamins (Table 2), and 10 mmol / L sodium acetate, with deionized water as the solvent. Sodium acetate was the sole carbon source of the medium. After preparation, the solution was aerated with nitrogen for 20 min. In this embodiment, the inoculation amount of natural environmental sediment sludge in each reactor is approximately 50 mg / L.

[0030] Table 1. Mineral solution formulations:

[0031]

[0032] Table 2 Vitamin Solution Formulas:

[0033]

[0034] Four reaction apparatuses were constructed to cultivate mature biofilms under different conditions: ① QS experimental group: A typical N-acyl homoserine lactone (AHLs), namely N-(3-oxododecyl)-l-homoserine lactone (3OC12-HSL), was added to the culture system to 10 μmol / L as an exogenous QS signaling molecule to stimulate the cultured biofilm; ② AC experimental group: Aspergillus acylase AC (Acylase), a commonly used drug that specifically quenches exogenous and endogenous QS signals (AHLs), was added to the culture system to a final concentration of 6 μg / mL; ③ QS-AC experimental group: The exogenous QS signaling molecule N-(3-oxododecyl)-l-homoserine lactone (3OC12-HSL) (10 μmol / L) and aspergillus acylase AC (6 μg / mL) were added to the culture system simultaneously; ④ Blank control group. In this embodiment, the biofilm culture system typically yields a mature and stable biofilm on the carbon-based material after approximately 25 days of cultivation at room temperature (25°C–37°C). Unless otherwise stated, all biofilm tests conducted in parallel reactors of this invention use mature and stable biofilm samples.

[0035] After the biofilm matured, the carbon-based material was removed, and a 0.3cm × 2.5cm × 0.1cm piece of carbon-based material with the biofilm structure was gently cut along the edge. The carbon-based material was placed in a 1.5mL centrifuge tube, and 1mL of 1×PBS buffer solution (pH=7) was added to ensure complete immersion. First, 1μL of Thermo LIVE / DEAD BacLight (catalog number: L-7012) fluorescent dye solution A was added, followed by 1μL of solution B. The mixture was stirred and allowed to stand for 20 minutes. The glass slide was wiped with an alcohol swab, and after the liquid on the slide surface had completely evaporated, the carbon-based material was gently placed on the slide. Immediately afterward, laser confocal electron microscopy (CLSM) was used to visually characterize the biofilm morphology. The imaging results show (e.g.) Figure 1 Compared to the other three groups, the QS experimental group had the highest proportion of live cells in its biofilm, and the presence of QS signaling molecules made the biofilm thicker and denser.

[0036] Example 2:

[0037] QS signaling molecule-regulated cultured biofilm for treating catering wastewater with a salt concentration of 10% (mass fraction):

[0038] The biofilm culture system consisted of a nutrient culture medium (same as in Example 1), carbon-based material (same as in Example 1, 2.5cm × 3cm × 0.1cm), and activated sludge from an urban wastewater treatment system (from Hongsen Environmental Protection Technology Co., Ltd., with an inoculum concentration of 50mg / L), and was carried out under anaerobic conditions (same as in Example 1). The activated sludge from the urban wastewater treatment system served as the microbial inoculum, and the biofilm formed on the carbon-based material.

[0039] Two reaction devices, a QS experimental group and a blank control group, were constructed according to Example 1. Mature biofilms were obtained by culturing under ambient temperature conditions. Carbon-based materials containing mature biofilms from each group were collected and used to treat 100 mL of catering wastewater with a salt concentration of approximately 10% (the main component of the salt is NaCl) for 4 hours at ambient temperature. The wastewater quality parameters were: COD 1000-1200 mg / L, temperature 28℃, ammonia nitrogen 0.5 g / L, pH 6.8, conductivity 4.91 mS / cm, and total phosphorus 1.39 g / L. 1 mL of the system reaction solution was collected at 0, 6, 18, 30, 42, 54, and 66 hours, and the COD content was determined using the potassium dichromate method. The results are as follows: Figure 2 As shown, the COD consumption rate in the QS experimental group was much higher than that in the blank control group, proving that the QS signaling molecule effectively improved biofilm activity and promoted COD degradation.

[0040] Example 3:

[0041] The protection of biomembrane electroactivity by QS signaling molecules:

[0042] Taking laboratory conditions as an example, a typical three-electrode system or a typical microbial electrochemical system was constructed. Microorganisms are more likely to form biofilms at the anodic potential. Firstly, to facilitate collection, characterization, and comparison of biofilm differences, a graphite plate (3cm × 2.5cm × 0.1cm) was artificially prepared as the working electrode (anodic potential). Its surface was polished with sandpaper (grit type 400), and then immersed in 3mol / L HNO3 solution for 12 hours to improve hydrophilicity. After removal, it was rinsed thoroughly with water and dried in an oven at 60℃. A hole was drilled in the center of the edge of the graphite plate using a 1.1mm diameter twist drill bit, and then titanium wire was threaded through the hole and fixed in place. The graphite plate was placed vertically at the bottom of the reactor, but not in contact with the bottom of the flask. A constant voltage of 0.2V was continuously applied to the working electrode using a potentiometer. Ag / AgCl was used as the reference electrode, with a potential of 0.2V applied. The probe was placed below the surface of the nutrient culture medium, and a 0.5mm diameter titanium wire was wound around the reference electrode as the counter electrode. A schematic diagram of the reaction device structure of the microbial electrochemical system is shown below. Figure 3 Natural environmental sediment sludge was used as the inoculum for the microorganisms in the microbial fuel cell, and the culture temperature was 25℃~37℃. The natural environmental sediment sludge was obtained by dredging from 10~20cm below the water surface of a long-running sediment microbial fuel cell and was used as the reactor inoculum, with an inoculum concentration of 50mg / L in each reactor.

[0043] Following the steps in Example 1, the system culture medium was 100 mL of nutrient medium. Four reaction devices were constructed: a QS experimental group, an AC experimental group, a QS-AC experimental group, and a blank control group. Different conditions were set up to acclimate and cultivate mature biofilms (EABs). A three-electrode reaction device was connected to an electrochemical workstation (CHI 1040c) for periodic cultivation. When the system current reached its peak, the EABs were considered mature and stable, and reproducible over at least two consecutive cycles. Generally, after four cycles (approximately 25 days) of cultivation, mature and stable biofilms were obtained on the graphite plates. The current density changes over time during biofilm formation in the four reaction devices were continuously recorded using the time-of-flight ample method (it curve) on the electrochemical workstation (CHI 1040c). Figure 4 The current density varied under different culture conditions, with the QS experimental group exhibiting the strongest biofilm current density, exceeding 0.3 mA / cm². 2 The group with added QS quencher (AC experimental group) showed significantly lower current density. This demonstrates that quorum sensing molecules have a positive regulatory effect on the enhancement of biological membranes, and the addition of QS signaling molecules helps to improve biological membrane stability.

[0044] Four groups of culture devices formed biofilms on graphite plates. Mature biofilms were generally obtained after approximately 25 days of cultivation. The mature EABs graphite plates were then removed and completely immersed in a 10% NaCl solution (in Example 1, the nutrient medium did not contain sodium acetate but instead contained 10% sodium chloride) for 4 hours to simulate a high-salt shock. After removal, they were placed back into the reactor, and new nutrient medium (same as in Example 1) was added. The first current density cycle of the biofilm was recorded in real time using the time-and-time curve (it curve) on an electrochemical workstation. The parameters for the current-time curve were set as follows: constant voltage 0.2V, sampling interval 90s, rest time 0s, and sampling time 10 seconds. 6 s, sensitivity 0.01 A / V, scan number 1. Results are as follows: Figure 5 As shown, extreme salt solution shocks led to a decrease in current density and a reduction in biofilm in all groups. The current density in the blank control group and the AC experimental group decreased significantly (70.0% and 93.3%, respectively). The QS experimental group showed the smallest decrease in current density, at 50.7%, with the highest current density reaching 0.17 mA / cm². 2 This directly proves that QS signaling has an indirect effect on biofilms under high-salt conditions. Although the biofilm current density will inevitably be affected by high salt shock, QS signaling molecules can significantly improve the salt tolerance of EABs.

[0045] The diagram illustrates the change in current density over time of the mature biofilms formed in the four culture devices in saline wastewater (10% salinity). Figure 6As shown, the salt resistance of carbon-based materials containing biofilms is long-lasting, and subsequent experiments have proven that it can be maintained for 1-2 months.

[0046] Example 4:

[0047] QS signaling molecules regulate the culture of biofilms for municipal wastewater treatment:

[0048] The biofilm culture system consisted of 100 mL of nutrient medium (same as in Example 1), carbon-based material (same as in Example 1, 2.5 cm × 3 cm × 0.1 cm), and activated sludge from an urban wastewater treatment system (same as in Example 2, inoculum size 50 mg / L), and was carried out under anaerobic conditions (same as in Example 1). The activated sludge from the urban wastewater treatment system served as the microbial inoculum, and the biofilm formed on the carbon-based material.

[0049] Two reaction devices, a QS experimental group and a blank control group, were constructed according to Example 1. After obtaining a mature biofilm, the nutrient medium was replaced with 100 mL of municipal sewage (the main salt components are NaCl, CaCl2, and Na2SO4). The water quality parameters were: COD 500–600 mg / L, temperature 23℃, ammonia nitrogen 0.85 g / L, pH 6.5, conductivity 3.36 S / m, and total phosphorus 2.42 g / L. 1 mL of the system reaction solution was collected at 0, 6, 18, 30, 42, 54, and 66 hours, and the COD content was determined using the potassium dichromate method. The results are as follows: Figure 7 As shown, the COD consumption rate in the QS experimental group was higher than that in the blank control group, proving that the QS signaling molecule effectively improved biomembrane activity and promoted COD reduction.

Claims

1. A method for enhancing biofilm stability in a wastewater treatment system based on quorum sensing, characterized in that, The following steps are involved: N-acylhomoserine lactone, a quorum sensing signaling molecule, is added to a biofilm culture system to culture the biofilm. The N-acylhomoserine lactone is N-(3-oxododecyl)-l-homoserine lactone, and the concentration of the N-acylhomoserine lactone added is 5~15 μmol / L. The wastewater is wastewater with a mass fraction of 10% salt concentration.

2. The method according to claim 1, characterized in that, The biofilm culture system consists of a nutrient culture medium, carbon-based materials, and activated sludge. The nutrient culture medium formula is 0.31 g / L NH4Cl, 0.13 g / L KCl, 2.27 g / L NaH2PO4·2H2O, mineral solution, vitamins, and 10 mmol / L sodium acetate, with deionized water as the solvent.

3. The method according to claim 1, characterized in that, The quorum sensing signal molecules serve as regulatory factors that enhance microbial stability, and the biofilm is a biofilm system formed from the initiation stage to domestication maturity.

4. A biofilm obtained using the method described in any one of claims 1-3.

5. The application of the biofilm according to claim 4 in the treatment of sewage or wastewater.

6. The application according to claim 5, characterized in that, The wastewater or sewage is wastewater or sewage with a salt concentration of 10% by mass, and the effective period of the salt resistance of the biofilm is 1-2 months.

7. The application according to claim 6, characterized in that, The salt is one or more of NaCl, CaCl2, Na2SO4, and MgSO4.

8. The application according to claim 5, characterized in that, The treatment of wastewater or sewage includes the removal of organic pollutants, with a chemical oxygen demand (COD) load of 50~1000 mg / L. The wastewater originates from domestic water use, and the sewage originates from tailwater from food processing, metallurgy, or chemical industries.

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