A method for accelerating microbial biofilm formation in toxicity testing

Abstract

The present invention proposes a method for accelerating the biofilm formation of microorganisms in toxicity detection, comprising the following steps: placing an anode of a microbial fuel cell in an anode chamber containing a biofilm formation nutrient solution, and then inoculating microorganisms in the biofilm formation nutrient solution to form biofilms in stages: S1, the first stage, the culture time is 16 to 18 hours, and the formula of the biofilm formation nutrient solution is a culture medium: a volume ratio of a water sample to be detected of 2:1; S2, the second stage, the culture time is 16 to 18 hours, and the formula of the biofilm formation nutrient solution is a culture medium: a volume ratio of a water sample to be detected of 1:2; S3, the third stage, the culture time is 16 to 1 8h, the formula of biofilm nutrient solution is the test water sample; when biofilm is formed, the biofilm nutrient solution is intermittently circulated in the anode chamber by a peristaltic pump; after the previous stage, the peristaltic pump stops operating, stands for 2 to 3h, and then the supernatant is discharged, the discharge volume is 40% to 50% of the total volume of the biofilm nutrient solution, and then the biofilm nutrient solution of the next stage is injected to make up; the components of the culture medium include yeast extract powder, casein, potassium chloride, sodium citrate, magnesium sulfate, calcium chloride and signal molecules, and the pH value of the culture medium is 7.2 to 7.6; the signal molecule is one of c-di-GMP and AI-2. c-di-GMP and AI-2 in the culture medium of the present invention together constitute a complex signal molecule network, which coordinates the behavior, metabolism and biofilm formation of bacteria through a multi-level regulatory mechanism, improves the adaptability and passage efficiency of the population, and shortens the biofilm time.

Description

Technical Field

[0001] The invention relates to the technical field of sewage treatment, and in particular to a method for accelerating microbial biofilm formation in toxicity detection. Background Art

[0002] Microbial fuel cells (MFCs) are devices that use the catalytic action of microorganisms to convert chemical energy stored in organic matter into electrical energy. The device can monitor water quality by using the sensitivity of electricity-producing microorganisms to toxic substances. When toxic substances are present in the water, the activity of electricity-producing microorganisms is inhibited and the amount of electricity produced decreases. In this device, microorganisms need to attach and grow on the anode surface of the microbial fuel cell to form a biofilm in order to effectively transfer electrons. This process is called biofilm formation.

[0003] Existing biofilm formation methods usually take several weeks to form a stable and effective biofilm, so it is necessary to develop a method that shortens the biofilm formation time. Summary of the invention

[0004] In view of this, the present invention proposes a method for accelerating the biofilm formation of microorganisms in toxicity detection, which can achieve stable biofilm formation in 2 days with a large amount of biofilm formation.

[0005] The technical solution of the present invention is implemented as follows: The present invention provides a method for accelerating the formation of microbial biofilm in toxicity detection, comprising the following steps:

[0006] The anode of the microbial fuel cell is placed in the anode chamber containing biofilm nutrient solution, and then the microorganisms are inoculated into the biofilm nutrient solution to form biofilm in stages:

[0007] S1, the first stage, the culture time is 16-18h, and the formula of biofilm nutrient solution is culture medium: the volume ratio of the water sample to be tested is 2:1;

[0008] S2, the second stage, the culture time is 16-18h, and the formula of biofilm nutrient solution is culture medium: the volume ratio of the water sample to be tested is 1:2;

[0009] S3, the third stage, the culture time is 16-18h, and the biofilm nutrient solution formula is the test water sample;

[0010] When biofilm is forming, the biofilm nutrient solution circulates intermittently in the anode chamber through a peristaltic pump;

[0011] After the previous stage is finished, the peristaltic pump stops working, and the tube is left to stand for 2-3 hours, and then the supernatant is discharged, and the discharge volume is 40%-50% of the total volume of the biofilm nutrient solution, and then the biofilm nutrient solution of the next stage is injected to make up the volume;

[0012] The components of the culture medium include yeast extract powder, casein, potassium chloride, sodium citrate, magnesium sulfate, calcium chloride and signal molecules, and the pH value of the culture medium is 7.2-7.6;

[0013] The signal molecule is one of c-di-GMP and AI-2.

[0014] Specifically, c-di-GMP promotes the initial attachment of bacteria to the surface and the stable formation of biofilms by regulating the synthesis of adhesion factors (such as cellulose, polysaccharides, etc.). c-di-GMP can inhibit the synthesis or motility of bacterial flagella, thereby reducing the free state of bacteria, enhancing the attachment tendency and the formation of biofilms. As a second messenger, c-di-GMP activates a multi-level signal transmission network by binding to receptor proteins, coordinating the attachment, aggregation and maturation of bacteria and biofilms. AI-2 promotes the formation and stability of biofilms and increases the thickness and density of biofilms by regulating intercellular signal transmission and the synthesis of adhesion factors. AI-2 coordinates the aggregation and biofilm formation of different types of bacteria through cross-species signal transmission, thereby enhancing the diversity and stability of biofilms.

[0015] c-di-GMP and AI-2 work together to enhance the initial attachment of bacteria to the surface and the stability of the biofilm by regulating the expression of biofilm-related genes.

[0016] Based on the above technical scheme, preferably, the culture medium formula is: yeast extract powder 3-5 g / L, casein 2-4 g / L, potassium chloride 0.3-0.5 g / L, sodium citrate 0.05-0.1 g / L, magnesium sulfate 0.02-0.1 g / L, calcium chloride 0.05-0.1 g / L and signal molecule 1-2 mg / L.

[0017] On the basis of the above technical solution, preferably, the components of the culture medium further include rhamnolipid, and the amount of the rhamnolipid is 0.5-1 mg / L.

[0018] Specifically, rhamnolipid is a highly efficient surfactant that can significantly reduce the surface tension of liquids, allowing microorganisms to more easily attach to the anode of microbial fuel cells, thereby shortening the biofilm formation time. Secondly, rhamnolipid can promote microorganisms to produce more extracellular polymers (EPS), which form bridges between microorganisms, help cells adhere and aggregate, and thus accelerate the formation and maturation of biofilms.

[0019] On the basis of the above technical solution, preferably, the components of the culture medium further include glycerol glucoside, and the amount of glycerol glucoside is 0.5-1 mg / L.

[0020] Signal molecules will be degraded in liquid environments or inactivated when the chemical environment changes. Glycerol glucoside can protect these signal molecules from degradation through its stabilization effect, ensuring their continued effectiveness during the biofilm formation process. In addition, the presence of glycerol glucoside can change the surface charge or spatial conformation of signal molecules, making it easier for them to bind to receptors on target cells, thereby improving the efficiency of signal transmission.

[0021] On the basis of the above technical solution, preferably, the microorganism is Bacillus or Gemmatimonas.

[0022] On the basis of the above technical solution, preferably, the inoculation amount of the bacterial strain is 0.01wt% to 0.1wt%.

[0023] On the basis of the above technical solution, preferably, during the staged biofilm formation, the nutrient solution circulates for 0.5 to 1 hour every 6 to 9 hours.

[0024] Based on the above technical solution, preferably, the flow rate of the peristaltic pump in the first stage is 20-25 r / min, the flow rate of the peristaltic pump in the second stage is 10-15 r / min, and the flow rate of the peristaltic pump in the third stage is 5-8 r / min for biofilm formation.

[0025] In the process of microbial biofilm formation, the flow rate of the peristaltic pump is an important parameter that significantly affects the attachment, proliferation and biofilm formation of microorganisms. Maintaining a high flow rate in the first stage can increase the transfer rate of nutrients and dissolved oxygen in the culture medium, promote the initial attachment and rapid growth of microorganisms; secondly, a high flow rate can also be used to remove weakly attached microorganisms and selectively retain microorganisms with strong adhesion. Appropriately reducing the flow rate in the second stage helps to maintain the supply of nutrients and the discharge of metabolic waste, support the proliferation of microorganisms and increase the thickness of the biofilm. In the third stage, the low flow rate provides a relatively stable environment, which is conducive to the maturation of the biofilm and the formation of functional microbial communities, and maximizes the simulation of the nutritional status in the actual sample. A hydraulic retention time of 6 to 9 hours is set for each stage to ensure that the microorganisms have enough time for attachment, proliferation and biofilm maturation, and optimize the effect of the entire biofilm formation process.

[0026] On the basis of the above technical solution, preferably, the anode material of the microbial fuel cell is a carbon substrate.

[0027] On the basis of the above technical solution, preferably, the temperature during biofilm formation is 30-37°C.

[0028] Compared with the prior art, the method for accelerating microbial biofilm formation in a toxicity test of the present invention has the following advantages:

[0029] Beneficial effects:

[0030] (1) c-di-GMP and AI-2 work together to regulate the expression of biofilm-related genes, thereby enhancing the initial attachment of bacteria to the surface and the stability of the biofilm. Among them, c-di-GMP mainly acts on signal transduction and gene expression regulation within bacteria, while AI-2 plays a role in signal transduction and group behavior coordination between bacteria. c-di-GMP and AI-2 together constitute a complex signal molecule network, which coordinates bacterial behavior, metabolism and biofilm formation through multi-level regulatory mechanisms, improves group adaptability and generation efficiency, and shortens biofilm formation time. (2) Rhamnolipids reduce the surface tension of the liquid, allowing microorganisms to more easily attach to the anode of the microbial fuel cell. Secondly, rhamnolipids can promote microorganisms to produce more extracellular polymers, which helps cell adhesion and aggregation, thereby accelerating the formation and maturation of biofilms.

[0031] (3) Glycerol glucoside protects these signal molecules from degradation through its stabilization effect, ensuring their continued effectiveness during the biofilm formation process. In addition, the presence of glycerol glucoside can make it easier for them to bind to receptors on target cells, thereby enhancing the efficiency of signal transmission.

[0032] (4) The present invention can effectively control the attachment, proliferation and biofilm formation of microorganisms by adjusting the flow rate of the peristaltic pump in stages. The high flow rate in the initial stage is conducive to selective attachment and rapid growth, the medium flow rate in the middle stage promotes proliferation and stable growth of biofilm, and the low flow rate in the later stage ensures the maturity and stability of the biofilm. The hydraulic retention time of each stage is set to 6 to 9 hours to ensure that the microorganisms have enough time to complete the physiological processes of each stage, thereby optimizing the overall effect of microbial biofilm. DETAILED DESCRIPTION

[0033] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] The water sample to be detected in the present invention is domestic sewage or industrial wastewater, and the present embodiment uses sewage with a BOD concentration of 200 mg / L as the test water sample. The signal molecules used in the present invention are c-di-GMP and AI-2, both of which are purchased from Sigma-Aldrich Trading Co., Ltd.

[0035] The microorganisms in the embodiments of the present invention are all developed by our company. The bacillus selected is Nitrobacter sp. HZ-N-008, with a preservation number of CGMCC No. 30530; the Gemmatimonas selected is Bacillus velezensis DL-25, with a preservation number of CCTCC NO: M 20231209.

[0036] Example 1

[0037] The method for accelerating microbial biofilm formation in the toxicity detection of this embodiment comprises the following steps:

[0038] S1, preparing the first stage biofilm nutrient solution: mixing the culture medium and the water sample to be tested in a volume ratio of 2:1 (a total of 1 L), and then inoculating Nitrobacter (Nitrobacter sp.) HZ-N-008 into the biofilm nutrient solution at an inoculation rate of 0.1 wt%.

[0039] The anode (graphite rod) of the carbon-based microbial fuel cell was placed in the anode chamber (1.2 L, working volume 1 L), and then a peristaltic pump was used to pump the biofilm nutrient solution into the anode chamber at a flow rate of 20 r / min. The hydraulic retention time was 6 h. The peristaltic pump was started again to circulate and pump at a flow rate of 20 r / min for 0.5 h, and then circulate and pump for 0.5 h every 6 h. The cycle was repeated. The biofilm time in the first stage was 16 h and the temperature was 30°C.

[0040] S2, after the first stage, the peristaltic pump stops working, and the solution is allowed to settle for 2 hours. Then the supernatant is discharged, and the discharge volume is 50% of the total volume of the biofilm nutrient solution. Then the peristaltic pump is used to inject the biofilm nutrient solution of the second stage at a flow rate of 10r / min to make up to 1L. The hydraulic retention time is 6h. Then the peristaltic pump is used to circulate the solution at a flow rate of 10r / min for 0.5h, and then circulate the solution for 0.5h every 6h, and the cycle is repeated.

[0041] In the second stage, the biofilm nutrient solution is: the culture medium and the water sample to be tested are mixed in a volume ratio of 1:2, the biofilm formation time is 16 hours, and the temperature is 30°C.

[0042] S3, after the second stage, the peristaltic pump stops working, and the solution is allowed to settle for 3 hours. Then the supernatant is discharged, and the discharge volume is 50% of the total volume of the biofilm nutrient solution. Then the peristaltic pump is used to inject the biofilm nutrient solution of the second stage at a flow rate of 5r / min to make up to 1L. The hydraulic retention time is 6h. Then the peristaltic pump is used to circulate the solution at a flow rate of 5r / min for 0.5h, and then circulate the solution for 0.5h every 6h.

[0043] In the third stage, the biofilm nutrient solution is the test water sample, the biofilm formation time is 16 hours, and the temperature is 30℃.

[0044] The formulas of the culture medium for the above three stages are: yeast extract powder 4g / L, casein 3g / L, potassium chloride 0.4g / L, sodium citrate 0.08g / L, magnesium sulfate 0.06g / L, calcium chloride 0.07g / L, signal molecule c-di-GMP 1mg / L, pH 7.2.

[0045] Example 2

[0046] The difference between Example 2 and Example 1 is that 0.6 mg / L rhamnolipid is added to the formula of the culture medium, and the rest of the contents are the same.

[0047] Example 3

[0048] The difference between Example 3 and Example 2 is that 0.8 mg / L glycerol glucoside is added to the formula of the culture medium, and the rest of the contents are the same.

[0049] Example 4

[0050] The method for accelerating microbial biofilm formation in the toxicity detection of this embodiment comprises the following steps:

[0051] S1, preparing the first stage biofilm nutrient solution: the culture medium and the water sample to be tested are mixed in a volume ratio of 2:1 (a total of 1 L), and then Bacillus velezensis DL-25 is inoculated into the biofilm nutrient solution at an inoculation rate of 0.05wt%.

[0052] Place the anode of the microbial fuel cell (carbon-based graphite rod or carbon paper) in the anode chamber (1.2L, working volume 1L), and then use a peristaltic pump to pump the biofilm nutrient solution into the anode chamber at a flow rate of 23r / min. The hydraulic retention time is 8h. Start the peristaltic pump again and circulate the pump at a flow rate of 23r / min for 1h. Then circulate the pump for 1h every 8h, and repeat the cycle. The biofilm time in the first stage is 17h and the temperature is 37°C.

[0053] S2, after the first stage, the peristaltic pump stops working, and the solution is allowed to settle for 2 hours. Then the supernatant is discharged, and the discharge volume is 45% of the total volume of the biofilm nutrient solution. Then the peristaltic pump is used to inject the biofilm nutrient solution of the second stage at a flow rate of 15r / min to make up to 1L. The hydraulic retention time is 8h. Then the peristaltic pump is used to circulate the solution at a flow rate of 15r / min for 1h. Then, the peristaltic pump is used to circulate the solution for 1h every 8h, and the cycle is repeated.

[0054] In the second stage, the biofilm nutrient solution is: the culture medium and the water sample to be tested are mixed in a volume ratio of 1:2, the incubation time is 17 hours, and the temperature is 37°C.

[0055] S3, after the second stage, the peristaltic pump stops working, and the solution is allowed to settle for 2 hours, then the supernatant is discharged, and the discharge volume is 45% of the total volume of the biofilm nutrient solution. Then the peristaltic pump is used to inject the biofilm nutrient solution of the third stage at a flow rate of 8 r / min to make up to 1L. The hydraulic retention time is 8 hours, and then the peristaltic pump is used to circulate and pump for 1 hour at a flow rate of 8 r / min, and then circulate and pump for 1 hour every 8 hours.

[0056] In the third stage, the biofilm nutrient solution is the test water sample, the incubation time is 17 hours, and the temperature is 37°C.

[0057] The formulas of the culture medium for the above three stages are: yeast extract powder 3g / L, casein 4g / L, potassium chloride 0.3g / L, sodium citrate 0.1g / L, magnesium sulfate 0.02g / L, calcium chloride 0.1g / L, rhamnolipid 0.5mg / L, glycerol glucoside 1mg / L, signal molecule AI-21mg / L, pH 7.6.

[0058] Example 5

[0059] The method for accelerating microbial biofilm formation in the toxicity detection of this embodiment comprises the following steps:

[0060] S1, preparing the first stage biofilm nutrient solution: mixing the culture medium and the water sample to be tested in a volume ratio of 2:1 (a total of 1 L), and then inoculating Nitrobacter (Nitrobacter sp.) HZ-N-008 into the biofilm nutrient solution at an inoculation rate of 0.01 wt%.

[0061] Place the anode of the microbial fuel cell (carbon-based graphite rod or carbon paper) in the anode chamber (1.2L, working volume 1L), and then use a peristaltic pump to pump the biofilm nutrient solution into the anode chamber at a flow rate of 25r / min. The hydraulic retention time is 9h. Start the peristaltic pump again and circulate the pumping at a flow rate of 25r / min for 40min. Then circulate the pumping for 40min every 9h, and repeat the cycle. The first stage biofilm time is 18h and the temperature is 35°C.

[0062] S2, after the first stage, the peristaltic pump stops working, and the solution is allowed to settle for 2.5 hours. The supernatant is then discharged, and the amount of liquid discharged is 40% of the total volume of the biofilm nutrient solution. The peristaltic pump is then used to inject the biofilm nutrient solution of the second stage at a flow rate of 13 r / min to make up to 1L. The hydraulic retention time is 9 hours. The peristaltic pump is then used to circulate the solution at a flow rate of 13 r / min for 40 minutes. After that, the solution is circulated for 40 minutes every 9 hours, and the cycle is repeated.

[0063] In the second stage, the biofilm nutrient solution is: the culture medium and the water sample to be tested are mixed in a volume ratio of 1:2, the incubation time is 18 hours, and the temperature is 35°C.

[0064] S3, after the second stage, the peristaltic pump stops working, and the solution is allowed to settle for 2.5 hours. The supernatant is then discharged, and the amount of liquid discharged is 40% of the total volume of the biofilm nutrient solution. The peristaltic pump is then used to inject the biofilm nutrient solution of the third stage at a flow rate of 7 r / min to make up to 1L. The hydraulic retention time is 9 hours. The peristaltic pump is then used to circulate the solution for 40 minutes at a flow rate of 7 r / min, and then circulate the solution for 40 minutes every 9 hours.

[0065] In the third stage, the biofilm nutrient solution is the test water sample, the incubation time is 18 hours, and the temperature is 35°C.

[0066] The formulas of the culture medium for the above three stages are: yeast extract powder 5g / L, casein 2g / L, potassium chloride 0.5g / L, sodium citrate 0.05g / L, magnesium sulfate 0.1g / L, calcium chloride 0.05g / L, rhamnolipid 1mg / L, glycerol glucoside 0.5mg / L, signal molecule c-di-GMP 2mg / L, pH 7.5.

[0067] Example 6

[0068] The method for accelerating microbial biofilm formation in the toxicity detection of this embodiment comprises the following steps:

[0069] S1, preparing the first stage biofilm nutrient solution: the culture medium and the water sample to be tested are mixed in a volume ratio of 2:1 (a total of 1 L), and then Bacillus velezensis DL-25 is inoculated into the biofilm nutrient solution at an inoculation rate of 0.04wt%.

[0070] Place the anode of the microbial fuel cell (carbon-based graphite rod or carbon paper) in the anode chamber (1.2L, working volume 1L), and then use a peristaltic pump to pump the biofilm nutrient solution into the anode chamber at a flow rate of 22r / min. The hydraulic retention time is 7h. Start the peristaltic pump again and circulate the pump at a flow rate of 22r / min for 1h. Then circulate the pump for 1h every 7h, and repeat the cycle. The first stage biofilm time is 16h and the temperature is 30℃.

[0071] S2, after the first stage, the peristaltic pump stops working, and the solution is allowed to settle for 3 hours. Then the supernatant is discharged, and the discharge volume is 50% of the total volume of the biofilm nutrient solution. Then the peristaltic pump is used to inject the biofilm nutrient solution of the second stage at a flow rate of 13r / min to make up to 1L. The hydraulic retention time is 7h. Then the peristaltic pump is used to circulate the solution at a flow rate of 13r / min for 1h, and then circulate the solution for 1h every 7h, and the cycle is repeated.

[0072] In the second stage, the biofilm nutrient solution is: the culture medium and the water sample to be tested are mixed in a volume ratio of 1:2, the incubation time is 16 hours, and the temperature is 30°C.

[0073] S3, after the second stage, the peristaltic pump stops working, and the solution is allowed to settle for 2.5 hours. The supernatant is then discharged, and the amount of liquid discharged is 50% of the total volume of the biofilm nutrient solution. The peristaltic pump is then used to inject the biofilm nutrient solution of the third stage at a flow rate of 6 r / min to make up to 1L. The hydraulic retention time is 9 hours. The peristaltic pump is then used to circulate the solution at a flow rate of 6 r / min for 0.5 hours, and then circulate the solution for 0.5 hours every 9 hours.

[0074] In the third stage, the biofilm nutrient solution is the test water sample, the incubation time is 16 hours, and the temperature is 30°C.

[0075] The formulas of the culture medium for the above three stages are: yeast extract powder 4.5 g / L, casein 3.5 g / L, potassium chloride 0.45 g / L, sodium citrate 0.06 g / L, magnesium sulfate 0.08 g / L, calcium chloride 0.06 g / L, rhamnolipid 0.7 mg / L, glycerol glucoside 0.9 mg / L, signal molecule c-di-GMP 1 mg / L and AI-2 1 mg / L, pH 7.5.

[0076] Comparative Example 1

[0077] Compared with Example 1, the formula of the culture medium in Comparative Example 1 lacks the signal molecule c-di-GMP, and the rest of the contents are the same.

[0078] Comparative Example 2

[0079] Comparative Example 2 Compared with Example 1, the concentration of the signal molecule c-di-GMP in the culture medium is 3 mg / L, and the rest of the contents are the same.

[0080] Comparative Example 3

[0081] Compared with Example 2, the concentration of rhamnolipid in the culture medium of Comparative Example 3 exceeds the specified range, specifically 1.5 mg / L, and the rest of the contents are the same.

[0082] Comparative Example 4

[0083] Compared with Example 3, the concentration of glycerol glucoside in the culture medium of Comparative Example 4 exceeds the specified range, specifically 1.5, and the rest of the contents are the same.

[0084] Comparative Example 5

[0085] Compared with Example 1, in Comparative Example 5, during the staged biofilm formation, the nutrient solution was static and no peristaltic pump was used to circulate the water flow. The rest of the contents were the same.

[0086] Comparative Example 6

[0087] Compared with Example 1, in Comparative Example 6, during the staged biofilm formation, the flow rate of the peristaltic pump in the three stages was 20 r / min, and the rest of the contents were the same.

[0088] Construction of microbial fuel cell: 1L of biofilm nutrient solution was placed in the anode chamber of the microbial fuel cell, 0.1mol / L phosphate buffer was added to the cathode chamber, the anode chamber and cathode chamber of the microbial fuel cell were separated by a proton exchange membrane, the anode electrode was made of carbon felt (2.0cm×2.0cm, thickness of 0.3cm), the cathode electrode was made of platinum sheet (1.0cm×1.0cm), and a 1 ohm fixed resistor was connected to the external circuit. The fuel cell was maintained in operation; when the highest output voltage during the continuous biofilm formation period no longer increased, the biofilm formation was considered complete.

[0089] Biofilm detection method: The amount of biofilm was characterized by measuring protease using the Coomassie Brilliant Blue method, the thickness of the biofilm was characterized by laser confocal microscopy, and the biofilm formation efficiency was characterized by the reactor start-up time. The results are shown in Table 1.

[0090] Table 1 Biofilm Efficiency of Examples and Comparative Examples

[0091]

[0092]

[0093] In the comparative examples, biofilm formation was not completed within the time limit of the present invention, and therefore the time of each stage was appropriately extended, wherein each stage in comparative example 2 was extended to 48 h; and each stage in comparative examples 1 and 5 was extended to 20 h.

[0094] As shown in Table 1, biofilm formation can be completed in only 33 to 48 hours by using the method of the embodiments of the present invention, and biofilm formation can be achieved within 2 days. Among them, Example 6 has the best effect and the shortest biofilm formation time.

[0095] It can be seen from Examples 1 to 3 that the addition of rhamnolipids and glycerol glucoside to the culture medium, on the one hand, makes it easier for microorganisms to attach to the anode surface, and on the other hand, enhances the stability of the active substances of the signal molecules, protecting them from degradation and ensuring their continued effectiveness during the biofilm formation process; thereby achieving the technical effect of shortening the biofilm formation time and increasing the biofilm formation amount.

[0096] However, when the dosage of rhamnolipids and glycerol glucoside exceeds the specified range (see Comparative Examples 3 to 4), the opposite effect is achieved. This is because excessive rhamnolipids and glycerol glucoside will affect the signal transmission system of microorganisms, disrupt intercellular communication and synergy, and thus affect the formation process of the biofilm.

[0097] It can be seen from Example 1 and Comparative Example 1 that the addition of signal molecules c-di-GMP and AI-2 can significantly increase the biofilm formation speed of Bacillus or Bacillus, indicating that c-di-GMP and AI-2 can regulate signal transduction and gene expression in bacteria, thereby coordinating bacterial behavior, metabolism and biofilm formation, improving group adaptability and passage efficiency, and shortening biofilm formation time.

[0098] Comparative Example 2 shows that the more signal molecules are used, the better. When the amount exceeds the specified range, the biofilm amount is reduced and the biofilm time is prolonged. This is because the signal molecules exceeding the appropriate concentration will cause the signal transmission system to be overloaded, resulting in the inability of microbial cells to correctly perceive and respond to environmental signals, affecting the normal function of quorum sensing. In addition, excessive use of signal molecules will also interfere with the interaction between microorganisms and the environment, including inhibiting the normal activities of other microorganisms in the environment, leading to an imbalance in the ecosystem and affecting the diversity and stability of the microbial population.

[0099] Comparative Examples 5 to 6 show that static biofilm or continuous high-flow rate peristalsis will affect the amount of biofilm and biofilm time. This is because: the transmission of nutrients and metabolites under static conditions mainly depends on diffusion, which will cause the nutrients in the culture medium to be rapidly depleted around the microorganisms, and the local microenvironment (such as pH value, dissolved oxygen concentration, etc.) is easy to change, which is not conducive to the stable growth of microorganisms in the entire biofilm area. Static conditions easily lead to uneven local thickness of the biofilm, and such unevenness will affect the overall function and stability of the biofilm. Continuous high-flow rate peristalsis will produce a large shear force, which will cause the newly formed biofilm to be continuously washed and peeled off, making it difficult to stably adhere to the surface of the material, affecting the initial formation of the biofilm.

[0100] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for accelerating the formation of microbial biofilm in toxicity detection, characterized in that: The following steps are involved: The anode of the microbial fuel cell is placed in the anode chamber containing biofilm nutrient solution, and then the microorganisms are inoculated into the biofilm nutrient solution to form biofilm in stages: S1, the first stage, the culture time is 16-18h, and the formula of biofilm nutrient solution is culture medium: the volume ratio of the water sample to be tested is 2:1; S2, the second stage, the culture time is 16-18h, and the formula of biofilm nutrient solution is culture medium: the volume ratio of the water sample to be tested is 1:2; S3, the third stage, the culture time is 16-18h, and the biofilm nutrient solution formula is the test water sample; When biofilm is formed, the biofilm nutrient solution is intermittently circulated in the anode chamber through a peristaltic pump; after the previous stage, the peristaltic pump stops working, stands for 2 to 3 hours, and then the supernatant is discharged, the discharge volume is 40% to 50% of the total volume of the biofilm nutrient solution, and then the biofilm nutrient solution of the next stage is injected to make up; The components of the culture medium include: 3-5 g / L yeast extract, 2-4 g / L casein, 0.3-0.5 g / L potassium chloride, 0.05-0.1 g / L sodium citrate, 0.02-0.1 g / L magnesium sulfate, 0.05-0.1 g / L calcium chloride and 1-2 mg / L signal molecule; the pH value of the culture medium is 7.2-7.6; The signal molecule is one of c-di-GMP and AI-2; The components of the culture medium also include rhamnolipid and glycerol glucose, wherein the amount of rhamnolipid is 0.5-1 mg / L, and the amount of glycerol glucoside is 0.5-1 mg / L; The microorganism is bacillus or genomiconads, and the inoculation amount of the strain is 0.01wt% to 0.1wt%; During the staged biofilm formation period, the biofilm nutrient solution circulates for 0.5 to 1 hour every 6 to 9 hours; the flow rate of the peristaltic pump in the first stage is 20 to 25 r / min, the flow rate of the peristaltic pump in the second stage is 10 to 15 r / min, and the flow rate of the peristaltic pump in the third stage is 5 to 8 r / min; The anode material of the microbial fuel cell is a carbon substrate; The temperature during film formation is 30-37°C.

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