Method for realizing long-acting stable sensing by using NO to control thickness of electroactive biological membrane
By adding DETA NOATE to the electroactive biofilm culture medium to release NO, the biofilm thickness is regulated, and the sensor instability caused by electroactive biofilm thickening is solved, achieving long-term stable operation of the sensor and sustainability of signal output.
Patent Information
- Application Number
- CN202510739671.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-04
- Publication Date
- 2025-09-02
AI Technical Summary
Electroactive biofilms are prone to thickening during long-term operation, resulting in extended electron transfer paths and intensified diffusion resistance, affecting signal output stability and pollutant response sensitivity, making it difficult to achieve long-term stable sensing.
By adding DETA NONOate to the culture medium as the NO donor, NO is released to inhibit biofilm thickening, and the microbial signal conduction system is used to regulate the biofilm thickness to maintain the biofilm thickness in the range of 40-80 μm, and coordinate the sensing current.
Effectively control the thickness of electroactive biofilm, improve the adaptability and stability of the sensor in complex wastewater environments, extend the service life, avoid interference with the metabolic activity of electroproducing bacteria, and have good biocompatibility and environmental friendliness.
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Figure CN120574675A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of microbial electrochemical sensing technology, and particularly relates to a method for achieving long-term stable sensing by controlling the thickness of electroactive biofilm using nitric oxide (NO), aiming to achieve long-term stable sensing of pollutants in sewage. Background Art
[0002] Indicators closely related to biological effects, such as toxic pollutant concentrations and the biodegradability of organic matter, are crucial for regulating wastewater treatment processes and managing ecological risks. However, conventional physical and chemical methods struggle to reflect the biological effects of pollutants. Traditional biological detection methods (such as BOD5 and luminescent bacteria toxicity tests) suffer from delayed response, poor interference resistance, and high costs, making them difficult to meet the practical needs for sensitive, rapid, and online monitoring of pollutants in wastewater.
[0003] Microbial electrochemical sensors, an emerging technology, offer a solution to these challenges. Based on electroactive biofilms, these sensors directly convert microbial metabolic activity into measurable electrical signals through extracellular electron transfer by electrogenic bacteria, enabling real-time responses to pollutant concentrations. These sensors offer advantages such as simple operation, rapid response, and low cost, and have become a key research area for online wastewater quality monitoring.
[0004] However, during long-term operation, the electroactive biofilm, as the core sensing element, tends to thicken over time, resulting in an extension of the electron transfer path and an increase in the diffusion resistance within the membrane, which in turn causes a decrease in electroactivity. This not only affects the stability of the signal output, but also weakens the sensor's response sensitivity to pollutant fluctuations, becoming a key bottleneck restricting its long-term stable operation. Therefore, there is an urgent need for a method that can regulate the thickness of the electroactive biofilm and maintain the coordinated stability of the membrane structure and function to improve the long-term performance of the sensor. NO has been shown to optimize the three-dimensional structure of the biofilm and achieve controllable thinning of the membrane thickness by regulating microbial signal transduction systems (such as c-di-GMP), inducing the expression of dispersion-related genes, and inducing mild oxidative stress. Summary of the Invention
[0005] In order to overcome the shortcomings of the above-mentioned prior art, the purpose of the present invention is to provide a method for achieving long-term stable sensing by controlling the thickness of electroactive biofilm using NO. This method can maintain a thin layer structure of the biofilm that can efficiently carry out extracellular electron transfer while maintaining the metabolic activity of electrogenic bacteria, thereby achieving long-term stability of sensing performance, and has practical application value in the field of sewage water quality monitoring.
[0006] In order to achieve the above object, the technical solution adopted by the present invention is:
[0007] A method for achieving long-term stable sensing by controlling the thickness of electroactive biofilms using NO. After pre-cultivation of the electroactive biofilm in a culture medium, DETA NONOate is added to the culture medium as an NO donor to release NO to inhibit the continued thickening of the biofilm, thereby achieving synergistic stability of the biofilm thickness and the sensing current.
[0008] In one embodiment, the electroactive biofilm pre-cultivation in the culture medium is achieved as follows:
[0009] Step 1: Using a phosphate buffer solution containing a carbon source as a culture medium, constructing an electroactive biofilm culture system in an anaerobic sealed environment;
[0010] Step 2: Inoculate the bacterial precipitate after centrifugation of domestic sewage into the culture system, and apply a constant voltage between the anode and cathode to form an electroactive biofilm; continuously monitor the sensing current signal, complete a culture cycle when the current is lower than the set threshold, and replace the culture medium. After several culture cycles, the electroactive biofilm pre-culture is completed.
[0011] In one embodiment, the electroactive biofilm culture system in an anaerobic sealed environment is constructed by the following method:
[0012] A closed reactor is used, equipped with anaerobic sealing accessories, and an electrode assembly is set in the reactor, including a carbon-based material anode and a stainless steel mesh cathode of the same size; a phosphate buffer solution (PBS) containing a carbon source is injected into the reactor, and dissolved oxygen is removed by nitrogen aeration.
[0013] In one embodiment, the reactor is one of a plexiglass cube, a serum bottle or a blue-mouthed reagent bottle; the anaerobic sealing accessory is a butyl rubber plug; and the carbon-based material anode is one of a graphite sheet, a graphite rod or carbon felt.
[0014] In one embodiment, the set threshold is 0.1 mA.
[0015] In one embodiment, the pre-culture period of the electroactive biofilm is 2 to 3 culture periods.
[0016] In one embodiment, the constant voltage applied between the cathode and anode is 0.5-0.9V.
[0017] In one embodiment, the final concentration of DETA NONOate is 200-800 μM, and after the pre-culture is completed, the biofilm will still be cultured for a long time, so DETA NONOate is still added when the culture medium is replaced in each culture cycle.
[0018] In one embodiment, the donor releases NO to inhibit the continued thickening of the biofilm. Due to the periodic addition of the donor, NO is periodically released. NO acts as a signaling molecule to regulate the expression of genes involved in biofilm formation and dispersion, thereby inhibiting the continued thickening of the biofilm during the culture process, maintaining it within a thickness range of 40 to 80 μm.
[0019] The present invention also claims protection for an electroactive biofilm prepared by the method of the present invention, which has a thickness and a stable sensing performance, wherein the thickness is 40 to 80 μm and the coefficient of variation of the multi-cycle maximum sensing current is less than 5%.
[0020] Compared with the prior art, the present invention has the following beneficial effects:
[0021] (1) This invention takes the molecular mechanism of electroactive biofilm formation as its fundamental starting point and utilizes NO signaling to target and downregulate intracellular c-di-GMP levels, thereby inhibiting biofilm formation. This provides a method for fundamentally inhibiting the thickening of electroactive biofilms. Compared to existing mechanical scraping methods, this method has the advantages of being simple to operate, avoiding physical damage, and being long-lasting and effective.
[0022] (2) The biofilm thickening inhibitor used in the present invention is NO, a natural signaling molecule with excellent biocompatibility. Furthermore, using DETA NONOate as the NO donor, dynamic regulation of NO signal concentration is achieved through sustained release, effectively controlling the increase in electroactive biofilm thickness while also avoiding interference with the metabolic activity of electrogenic bacteria. Compared to existing biochemical methods for biofilm thinning (such as fungicides), this method has the advantages of being environmentally friendly, highly controllable, and avoiding drug resistance.
[0023] (3) The present invention addresses the problems of decreased electron transfer efficiency and restricted material diffusion caused by excessive thickening of electroactive biofilms. By maintaining a thin layer structure through a thickness control strategy, the present invention effectively ensures the continuous stability of the electrical signal output. This not only improves the adaptability and stability of electroactive biofilms in complex sewage environments, but also significantly extends the service life of the sensor. In addition, the thickness control strategy has good versatility and is applicable to various online pollutant monitoring systems based on microbial electrochemical sensors, with broad engineering application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 The laser confocal microscopy three-dimensional image (A) and current density map (B) of the electroactive biofilm cultured using the method of the present invention. A constant voltage of 0.6 V was applied, and after three cycles of pre-culture, a NO donor (DETANONOate) was added at a final concentration of 500 μM.
[0025] Figure 2The laser confocal microscopy three-dimensional image (A) and current density map (B) of the electroactive biofilm cultured using the method of the present invention. A constant voltage of 0.7 V was applied, and after two cycles of pre-culture, a NO donor (DETANONOate) was added at a final concentration of 600 μM.
[0026] Figure 3 The laser confocal microscopy three-dimensional image (A) and current density map (B) of the electroactive biofilm cultured using the method of the present invention. A constant voltage of 0.7 V was applied, and after two cycles of pre-culture, a NO donor (DETANONOate) was added at a final concentration of 300 μM.
[0027] Figure 4 The laser confocal microscopy three-dimensional image (A) and current density map (B) of the electroactive biofilm cultured using the method of the present invention. A constant voltage of 0.8 V was applied, and after three cycles of pre-culture, a NO donor (DETANONOate) was added at a final concentration of 400 μM.
[0028] Figure 5 The BOD value of 0.5 g / L acetate simulated sewage was detected at three different time periods during electroactive biofilm cultivation.
[0029] Figure 6 To detect the current decay value of 0.2 mg / L tetracycline poison in sewage at three different time periods of electroactive biofilm cultivation. DETAILED DESCRIPTION
[0030] In order to explain the present invention in detail, the present invention will be further described below in conjunction with the accompanying drawings and specific examples. It should be understood by those skilled in the art that the content described below is illustrative and non-restrictive and should not be used to limit the scope of the present invention. Unless otherwise indicated, the specific experimental conditions or methods in the following examples are carried out according to the conventional conditions or methods in this field.
[0031] As mentioned above, in the prior art, the uncontrollable thickness of electroactive biofilms during long-term cultivation can lead to signal drift. Therefore, the present invention provides a method for controlling the thickness of electroactive biofilms mediated by NO signals to ensure sensing stability during long-term operation, specifically comprising the following steps:
[0032] (1) Construction of electroactive biofilm culture system.
[0033] In an embodiment of the present invention, the culture system mainly consists of a reactor, electrodes, and culture medium. The present invention requires the construction of an anaerobic sealed environment. Therefore, a sealed reactor (such as a plexiglass cube, serum bottle, blue-mouthed reagent bottle, etc.) is used, equipped with anaerobic sealing accessories such as butyl rubber stoppers; an electrode assembly is set in the reactor, including a carbon-based material anode (such as a graphite sheet, graphite rod, carbon felt, etc.) and a stainless steel mesh cathode, and the cathode and cathode are preferably of the same size; a phosphate buffer solution (PBS) containing a carbon source is injected into the reactor, and dissolved oxygen is removed by nitrogen aeration.
[0034] (2) Formation of electroactive biofilm.
[0035] The bacterial precipitate obtained after centrifugal treatment of domestic sewage is used as the inoculation source and inoculated into the culture system described in step (1). A constant voltage is applied between the anode and cathode to form an electroactive biofilm. For example, the constant voltage may preferably be 0.5 to 0.9 V. The sensing current signal is continuously monitored. When the current is lower than the set threshold (such as 0.1 mA), it is considered that a culture cycle has ended and fresh culture medium needs to be replaced to supplement the carbon source. After several culture cycles, the electroactive biofilm pre-culture is completed. For example, the pre-culture can be completed after 2 to 3 cycles of culture.
[0036] (3) Synergistic stabilization of electroactive biofilm thickness and sensing performance.
[0037] Pre-cultivation allows the biofilm to reach a certain thickness, at which electroactive biofilms exhibit better performance. Subsequently, the biofilm requires continued periodic cultivation, which can lead to continued thickening. To prevent this thickening, the present invention incorporates an NO donor during each subsequent long-term culture, adding it to the medium during each cycle. This prevents the biofilm from continuing to thicken, maintaining it at 40-80 μm over the long term, thus enabling long-term biofilm sensing.
[0038] Specifically, after pre-cultivation of the electroactive biofilm, DETA NONOate is added to the culture medium as a NO donor for long-term incubation. During this process, NO is released and inhibits biofilm thickening, leading to synergistic stabilization of biofilm thickness and sensing current. The final donor concentration is preferably 200-800 μM. The electroactive biofilm ultimately stabilizes within a thickness range of 40-80 μm, with a coefficient of variation of the maximum sensing current of less than 5% over multiple cycles, demonstrating excellent structural controllability and signal reproducibility.
[0039] DETA NONOate, a NO donor in this invention with the CAS number 146724-94-9, offers advantages such as a long half-life and slow NO release. While maintaining its inhibitory effect on electroactive biofilms, it minimizes the impact on the metabolic activity of electrogenic bacteria, ensuring stable sensor current signal output during long-term operation. In this invention, the final concentration refers to the final concentration of the NO donor in the culture medium after addition.
[0040] The principle of using NO to control the thickness of electroactive biofilms in the present invention is that NO, as a signal molecule, can regulate microbial signal transduction systems (such as c-di-GMP), induce the expression of dispersion-related genes, and trigger mild oxidative stress, thereby promoting the optimization of the three-dimensional structure of the biofilm and achieving control of the membrane thickness.
[0041] Furthermore, using DETA NONOate as a donor, it can slowly release NO, thereby achieving dynamic regulation of NO signal concentration through sustained release, ultimately effectively controlling the increase in the thickness of the electroactive biofilm while avoiding interfering with the metabolic activity of the electrogenic bacteria.
[0042] The following are several specific embodiments of the present invention.
[0043] Example 1
[0044] This embodiment provides a method for controlling the thickness of an electroactive biofilm using NO to achieve long-term stable output of a sensing current.
[0045] The culture system consists of a plexiglass cubic reactor, a graphite sheet anode, a stainless steel mesh cathode, and a culture medium. The bacterial precipitate obtained after centrifugal treatment of domestic sewage is used as the inoculation source. A constant voltage of 0.6V is applied between the anode and cathode to culture the electroactive biofilm. After three cycles of pre-culture, DETA NONOate with a final concentration of 500μM is added to the culture medium as an NO donor to release NO to inhibit the continued thickening of the biofilm. After multiple cycles of continuous culture, combined with laser confocal microscopy imaging and LIVE / DEAD BacLight Bacterial Viability Kits staining, it was observed that the thickness of the electroactive biofilm cultured using the method of the present invention was stable at 48μm ( Figure 1 A), the maximum sensing current is stable at 2.59±0.10mA( Figure 1 (B) The coefficient of variation is 3.7%, indicating excellent structural controllability and stable signal reproducibility.
[0046] Example 2
[0047] This embodiment provides a method for controlling the thickness of an electroactive biofilm using NO to achieve long-term stable output of a sensing current.
[0048] The culture system consists of an anaerobic serum bottle, a graphite sheet anode, a stainless steel mesh cathode, and a culture medium. The bacterial precipitate obtained after centrifugal treatment of domestic sewage is used as the inoculation source. A constant voltage of 0.7V is applied between the anode and cathode to culture the electroactive biofilm. After two cycles of pre-culture, DETA NONOate with a final concentration of 600μM is added to the culture medium as an NO donor to release NO to inhibit the continued thickening of the biofilm. After multiple cycles of continuous culture, combined with laser confocal microscopy imaging and LIVE / DEAD BacLight Bacterial Viability Kits staining, it was observed that the thickness of the electroactive biofilm cultured using the method of the present invention was stable at 58μm ( Figure 2 A), the maximum sensing current of multiple cycles is stable at 2.99±0.12mA( Figure 2 (B) The coefficient of variation is 4.1%, indicating excellent structural controllability and stable signal reproducibility.
[0049] Example 3
[0050] This embodiment provides a method for controlling the thickness of an electroactive biofilm using NO to achieve long-term stable output of a sensing current.
[0051] The culture system consists of an anaerobic serum bottle, a graphite rod anode, a stainless steel mesh cathode, and a culture medium. The bacterial precipitate obtained after centrifugal treatment of domestic sewage is used as the inoculation source. A constant voltage of 0.7V is applied between the anode and cathode to culture the electroactive biofilm. After two cycles of pre-culture, DETA NONOate with a final concentration of 300μM is added to the culture medium as an NO donor to release NO to inhibit the continued thickening of the biofilm. After multiple cycles of continuous culture, combined with laser confocal microscopy imaging and LIVE / DEAD BacLight Bacterial Viability Kits staining, it was observed that the thickness of the electroactive biofilm cultured using the method of the present invention was stable at 62μm ( Figure 3 The maximum sensing current of multiple cycles is stable at 3.20±0.13mA ( Figure 3 (B) The coefficient of variation is 4.0%, indicating excellent structural controllability and stable signal reproducibility.
[0052] Example 4
[0053] This embodiment provides a method for controlling the thickness of an electroactive biofilm using NO to achieve long-term stable output of a sensing current.
[0054] The culture system consists of a plexiglass cube, a carbon felt anode, a stainless steel mesh cathode, and a culture medium. The bacterial precipitate obtained after centrifugal treatment of domestic sewage is used as the inoculation source. A constant voltage of 0.8V is applied between the anode and cathode to culture the electroactive biofilm. After three cycles of pre-culture, DETA NONOate with a final concentration of 400μM is added to the culture medium as an NO donor to release NO to inhibit the continued thickening of the biofilm. After multiple cycles of continuous culture, combined with laser confocal microscopy imaging and LIVE / DEAD BacLight Bacterial Viability Kits staining, it was observed that the thickness of the electroactive biofilm cultured using the method of the present invention was stable at 75μm ( Figure 4 A), the maximum sensing current of multiple cycles is stable at 4.10±0.10mA( Figure 4 (B) The coefficient of variation is 2.4%, indicating excellent structural controllability and stable signal reproducibility.
[0055] Example 5
[0056] This embodiment provides a method for controlling the thickness of an electroactive biofilm using NO, and its application in detecting the stability of BOD in sewage.
[0057] The culture system consists of a plexiglass cube, a graphite sheet anode, a stainless steel mesh cathode and a culture solution. The bacterial precipitate obtained after centrifugal treatment of domestic sewage is used as the inoculation source, and a constant voltage of 0.7V is applied between the anode and the cathode to culture the electroactive biofilm. After two cycles of pre-culture, DETA NONOate with a final concentration of 400μM was added to the culture solution as a NO donor to release NO to inhibit the continued thickening of the biofilm. During the continuous culture period, a solution containing 0.5g / L sodium acetate was used as simulated sewage, and three BOD sensing tests were carried out at different time periods. The specific method is to add the simulated sewage as a culture solution to the reaction system, record the current changes, and perform classical coulomb calculations based on this to obtain the BOD content in the sewage. Figure 5 As shown, the results of three BOD sensing tests were stable at 250 mg / L, indicating that the electroactive biofilm cultured using the method of the present invention can achieve long-term and stable sensing of sewage BOD.
[0058] Example 6
[0059] This embodiment provides a method for controlling the thickness of an electroactive biofilm using NO, and its application in stable sensing and detection of toxic substances in sewage.
[0060] The culture system consists of an anaerobic serum bottle, a graphite sheet anode, a stainless steel mesh cathode and a culture medium. The bacterial precipitate obtained after centrifugal treatment of domestic sewage is used as the inoculation source. A constant voltage of 0.7V is applied between the anode and cathode to culture the electroactive biofilm. After two cycles of pre-culture, DETA NONOate with a final concentration of 500μM was added to the culture medium as a NO donor to release NO to inhibit the continued thickening of the biofilm. During the continuous culture period, 0.2mg / L tetracycline was used to simulate sewage toxic substances, and three toxicity sensing tests were carried out at different time periods. The specific method is to add simulated sewage toxic substances to the reaction culture medium and record the current decay. As Figure 6 As shown, the decay current of the three toxicity sensing tests was stable at 0.71 mA, indicating that the electroactive biofilm cultured using the method of the present invention can achieve long-term and stable sensing of toxic substances in sewage.
[0061] The present invention innovatively utilizes the NO regulation strategy to synergistically optimize the structural stability and signal reproducibility of the electroactive biofilm, constructing a sensing element that can operate stably for a long time, and providing a low-maintenance, high-reliability biosensing solution for real-time monitoring of pollutants in complex water quality environments.
Claims
1. A method for achieving long-term stable sensing by controlling the thickness of electroactive biofilm using NO, characterized in that: After the electroactive biofilm was pre-cultured in the culture medium, DETA NONOate was added to the culture medium as a NO donor to release NO to inhibit the continuous thickening of the biofilm, thereby synergistically stabilizing the biofilm thickness and the sensing current.
2. The method for achieving long-term stable sensing by controlling the thickness of electroactive biofilm using NO according to claim 1, characterized in that: The electroactive biofilm pre-cultivation is completed in the culture medium, and the implementation method is as follows: Step 1: Using a phosphate buffer solution containing a carbon source as a culture medium, constructing an electroactive biofilm culture system in an anaerobic sealed environment; Step 2: Inoculate the bacterial precipitate after centrifugation of domestic sewage into the culture system, and apply a constant voltage between the anode and cathode to form an electroactive biofilm; continuously monitor the sensing current signal, complete a culture cycle when the current is lower than the set threshold, and replace the culture medium. After several culture cycles, the electroactive biofilm pre-culture is completed.
3. The method for achieving long-term stable sensing by controlling the thickness of electroactive biofilm using NO according to claim 2, characterized in that: The method for constructing the electroactive biofilm culture system in an anaerobic sealed environment is as follows: A closed reactor is used, equipped with anaerobic sealing accessories, and an electrode assembly is set in the reactor, including a carbon-based material anode and a stainless steel mesh cathode of the same size; a phosphate buffer solution (PBS) containing a carbon source is injected into the reactor, and dissolved oxygen is removed by nitrogen aeration.
4. The method for achieving long-term stable sensing by controlling the thickness of electroactive biofilm using NO according to claim 2, characterized in that: The threshold value is set to 0.1 mA, and the constant voltage applied between the anode and cathode is 0.5 to 0.9 V.
5. The method for achieving long-term stable sensing by controlling the thickness of electroactive biofilm using NO according to claim 2, characterized in that: The pre-culture period of the electroactive biofilm is 2 to 3 culture periods.
6. The method for achieving long-term stable sensing by controlling the thickness of electroactive biofilm using NO according to claim 2, 3, 4 or 5, characterized in that: The final concentration of DETA NONOate is 200-800 μM, and is added again when the culture medium is replaced in each culture cycle after the pre-culture is completed.
7. The method for achieving long-term stable sensing by controlling the thickness of electroactive biofilm using NO according to claim 1, characterized in that: The donor releases NO to inhibit the continuous thickening of the biofilm and control the thickness to be 40 to 80 μm.
8. An electroactive biofilm with synergistically stable thickness and sensing performance, characterized in that: The film is prepared by the method according to any one of claims 1 to 8, has a thickness of 40 to 80 μm, and a coefficient of variation of the maximum sensing current over multiple cycles is less than 5%.
9. Use of the electroactive biofilm with synergistically stable thickness and sensing performance as claimed in claim 8 in the stable detection of BOD in sewage.
10. Use of the electroactive biofilm with synergistically stable thickness and sensing performance as claimed in claim 8 in the stable sensing detection of toxic substances in sewage.