Method for detecting concentration of acetic acid in anaerobic fermentation system by using bioelectrochemical sensor

Abstract

The invention discloses a method for detecting the concentration of acetic acid in an anaerobic fermentation system by using a bioelectrochemical sensor, acetic acid is detected by using the bioelectrochemical sensor, the bioelectrochemical sensor comprises a screen-printed electrode, and the working surface of the screen-printed electrode is modified with reduced graphene oxide and polymethylene blue. The working surface of the screen printing electrode is connected with acetokinase, pyruvate kinase and lactic dehydrogenase through a cross-linking agent. According to the method, the adopted sensor has better conductivity, higher response speed and higher current intensity, when the sensor is used for detecting the concentration of acetic acid in a fermentation system, the sensor has the advantages of being easy to operate, low in sample consumption, low in price, high in accurate measurement precision, high in detection speed and the like, and rapid detection of the concentration of acetic acid can be achieved; the acetic acid concentration in the anaerobic fermentation process in different periods can be timely and accurately obtained, effective data can be provided for anaerobic fermentation analysis, and the method has important significance for monitoring the process stability of anaerobic fermentation.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for detecting the concentration of acetic acid in an anaerobic fermentation system, and particularly relates to a method for detecting the concentration of acetic acid in an anaerobic fermentation system by using a bioelectrochemical sensor. BACKGROUND

[0002] Anaerobic fermentation is one of the important ways for waste resource utilization, but the fermentation process is affected by many factors such as the nature of the material, operating conditions, etc., and the process shows strong nonlinearity, variability and complexity. Volatile fatty acids as important intermediates can well reflect the degradation process of organic matter. If the concentration of volatile fatty acids is too low, the nutrients required by microorganisms are insufficient, which is not conducive to the subsequent methanogenesis process. If the concentration of volatile fatty acids is too high, it will lead to a decrease in pH, which inhibits the growth of microorganisms and is also not conducive to the subsequent process. When the VFA / alkalinity value is greater than 0.3-0.4, it indicates that the anaerobic fermentation is unstable. Studies have shown that acetic acid, as an important volatile fatty acid, its concentration reflects the total concentration of volatile fatty acids to some extent. The whole process of volatile fatty acids can be divided into four periods: adaptation period, start-up period, inhibition period, and recovery and stable period. In the adaptation period and start-up period, the concentration of acetic acid rises rapidly, only less than that of butyric acid. In the inhibition period, the concentration of acetic acid increases significantly, accounting for about 25% of the total volatile fatty acid concentration. In the recovery and stable period, the concentration of acetic acid gradually tends to be stable, and the percentage of the total volatile fatty acid concentration fluctuates in the range of 21-23%. Therefore, rapid detection of the concentration of acetic acid has a good indication effect on reflecting the total concentration of volatile fatty acids, and is of great significance for monitoring the process stability of anaerobic fermentation.

[0003] In recent years, traditional acetic acid quantitative measurement methods such as gas chromatography (GC) and high performance liquid chromatography (HPLC) are mostly complex and have a long detection period. The ultraviolet absorption method is an advanced method, but it depends on expensive instruments, skilled operators, etc. In addition, the electrochemical method has the advantages of simple process, convenient operation, low cost, short detection period, etc., so it has become one of the preferred methods for rapid detection of acetic acid concentration.

[0004] In the existing electrochemical method, the biological electrochemical sensor is used to react with acetic acid, and then the concentration of acetic acid is detected by detecting the current change in the reaction process. However, the existing electrochemical method still has defects such as narrow detection range and poor accuracy. The reason may be that the electron mediator is directly deposited on the surface of the electrode in the biological electrochemical sensor, which makes the conductivity between the electron mediator and the electrode poor, resulting in slow response of the biological electrochemical sensor and small response current, so that it is difficult to effectively collect the change of the current signal in the detection system, and finally leads to defects such as narrow detection range and poor accuracy. Especially, when the concentration of acetic acid in the system is high, it is more difficult to accurately collect the current signal, so it is more difficult to obtain accurate concentration data. Therefore, it is of great significance to obtain a biological electrochemical sensor with good conductivity, fast response speed and large current intensity for realizing accurate detection of the concentration of acetic acid in the anaerobic fermentation system. SUMMARY

[0005] The technical problem to be solved by the present application is to overcome the shortcomings of the prior art and provide a method for detecting the concentration of acetic acid in an anaerobic fermentation system by using a biological electrochemical sensor.

[0006] To solve the above technical problems, the present application adopts the following technical solutions:

[0007] A method for detecting the concentration of acetic acid in an anaerobic fermentation system by using a biological electrochemical sensor, the method is to detect acetic acid in the anaerobic fermentation liquid by using a biological electrochemical sensor; the biological electrochemical sensor comprises a working electrode; the working electrode is a screen-printed electrode; the working surface of the screen-printed electrode is modified with reduced graphene oxide, and the reduced graphene oxide is deposited with polymethylene blue; the working surface of the screen-printed electrode is connected with acetic acid kinase, pyruvate kinase and lactate dehydrogenase through a crosslinking agent.

[0008] The above method is further improved, and the crosslinking agent is polyethylene glycol diglycidyl ether.

[0009] The above method is further improved, and the preparation method of the working electrode comprises the following steps:

[0010] S1, dropping an oxidized graphene solution onto the working surface of a screen-printed electrode, drying to obtain an oxidized graphene modified screen-printed electrode;

[0011] S2, immersing the oxidized graphene modified screen-printed electrode in a methylene blue solution to carry out a polymerization reaction, to obtain a reduced graphene oxide and polymethylene blue modified screen-printed electrode;

[0012] S3. Acetylkinase, pyruvate kinase, lactate dehydrogenase and cross-linking agent are mixed and added to the surface of a screen-printed electrode modified with reduced graphene oxide and polymethylene blue. After drying, acetate kinase, pyruvate kinase and lactate dehydrogenase are loaded onto the screen-printed electrode modified with reduced graphene oxide and polymethylene blue to obtain the working electrode.

[0013] In a further improvement to the above method, in step S1, the graphene oxide solution is prepared by dispersing graphene oxide in water; the concentration of the graphene oxide solution is 0.1 mg / mL. -1 ~0.5mg mL -1 .

[0014] In a further improvement to the above method, in step S2, the methylene blue solution is prepared by dispersing methylene blue in water; the concentration of the methylene blue solution is 5 mM; the potential window during the polymerization reaction is -1 V to +1.2 V, and the scan rate is 10 mV s. -1 ~50mV s -1 The number of scans is 10 to 20.

[0015] In a further improvement to the above method, in step S2, the enzyme activity ratio of the acetate kinase, pyruvate kinase, and lactate dehydrogenase is 1:1 to 2:2 to 3; and the drying is carried out at a temperature of 4°C.

[0016] A further improvement to the above method, using a bioelectrochemical sensor to detect acetic acid in anaerobic fermentation broth, includes the following steps:

[0017] (1) Constructing a bioelectrochemical sensor;

[0018] (2) The anaerobic fermentation broth to be tested is added to the bioelectrochemical sensor for scanning, and the current of the anaerobic fermentation broth to be tested is collected.

[0019] (3) Input the current of the anaerobic fermentation broth to be tested into the standard linear regression equation of acetic acid concentration and current, and calculate the concentration of acetic acid in the anaerobic fermentation broth.

[0020] In a further improvement to the above method, in step (3), the standard linear regression equation between the acetic acid concentration and the current is:

[0021] y = 38.381 × x + 23.9167;

[0022] Where y is the current value of the anaerobic fermentation broth (nA), x is the acetic acid concentration in the anaerobic fermentation broth (mM), and R 2 The value is 0.99578, the linear range is 5mM to 17.5mM, and the detection limit is 2.5mM.

[0023] In a further improvement to the above method, step (2) involves using cyclic voltammetry to scan the anaerobic fermentation broth to be tested; the potential during the scanning process is -0.5V to +0.5V.

[0024] The above method is further improved in step (1), in which the construction method of the bioelectrochemical sensor includes the following steps: inserting the working electrode into the PBS buffer solution, adding phosphoenolpyruvate monopotassium salt, adenosine 5′-triphosphate disodium salt trihydrate, β-nicotinamide adenine dinucleotide reduced disodium salt hydrate, connecting to the electrochemical workstation, and obtaining the bioelectrochemical sensor.

[0025] Compared with the prior art, the advantages of the present invention are as follows:

[0026] (1) This invention provides a method for detecting the concentration of acetic acid in an anaerobic fermentation system using a bioelectrochemical sensor. The bioelectrochemical sensor is used to detect acetic acid in the anaerobic fermentation broth. The bioelectrochemical sensor includes a working electrode, which is a screen-printed electrode. The working surface of the screen-printed electrode is modified with reduced graphene oxide. Polymethylene blue is deposited on the reduced graphene oxide. Acetylkinase, pyruvate kinase and lactate dehydrogenase are connected to the working surface of the screen-printed electrode through a cross-linking agent. In this invention, reduced graphene oxide and polymethylene blue are modified on the working surface of a screen-printed electrode. Reduced graphene oxide enhances the electrode's conductivity, while polymethylene blue acts as an electron mediator. The combined modification of reduced graphene oxide and polymethylene blue on the working surface of the screen-printed electrode forms a film structure, further improving conductivity and increasing the enzyme loading area. This facilitates the loading of more functional enzymes onto the electrode surface. Furthermore, a cross-linking agent allows for the loading of more acetate kinase, pyruvate kinase, and lactate dehydrogenase onto the working surface of the screen-printed electrode. Through the combined action of the film structure and the cross-linking agent, acetate kinase, pyruvate kinase, and lactate dehydrogenase can be uniformly and firmly fixed onto the screen-printed electrode. The working surface of the electrode improves the stability of the bioelectrochemical sensor. Furthermore, the combined action of acetate kinase, pyruvate kinase, and lactate dehydrogenase converts acetic acid into lactic acid. Simultaneously, the oxidation current change during this conversion allows for the determination of a linear relationship between acetic acid concentration and current, which can then be used to determine the concentration of acetic acid in the anaerobic fermentation broth. In particular, compared to conventional enzyme sensors, the screen-printed electrode used in this invention has a surface loaded with more functional enzymes, enabling rapid conversion of acetic acid into lactic acid and rapid, accurate collection of the current signal in the system. Therefore, even when higher concentrations of acetic acid are present in the system, accurate detection of these higher concentrations is possible, resulting in better applicability and application prospects. Additionally, the use of a screen-printed electrode as the working electrode in this invention is convenient, portable, and inexpensive, thus reducing detection costs. In the method of this invention, the bioelectrochemical sensor, through the combined action of reduced graphene oxide and polymethylene blue, can significantly improve the conductivity of the electrode and load more functional enzymes, thereby enabling the sensor to have a faster response speed, a larger current intensity, and advantages such as high sensitivity, wide linear range, and low detection limit. When used to detect the acetic acid concentration in anaerobic fermentation systems, it has advantages such as simple operation, low sample consumption, low price, high precision, fast detection speed, and relatively simple sample pretreatment. It can realize the rapid detection of acetic acid concentration in the fermentation system and can timely and accurately obtain the acetic acid concentration at different stages of the anaerobic fermentation process, thus providing effective data for anaerobic fermentation analysis, which is of great significance for monitoring the process stability of anaerobic fermentation.

[0027] (2) In this invention, the screen-printed electrode modified with graphene oxide is immersed in a methylene blue solution for polymerization. Under cyclic voltammetry, methylene blue initially forms a free radical cation, which provides an electron to the graphene oxide. Then the graphene oxide is reduced. In subsequent cycles, it forms a polymer composite material through the covalent interaction between the simultaneously reduced graphene oxide and the oxidized methylene blue. Thus, the working surface of the screen-printed electrode is jointly modified with reduced graphene oxide and polymethylene blue, forming a film structure with a large specific surface area and good conductivity on the surface of the screen-printed electrode. This is beneficial to increasing the enzyme loading area and also to improving the conductivity of the electrode.

[0028] (3) In this invention, under the continuous reaction of acetic acid kinase (AK), pyruvate kinase (PK) and lactate dehydrogenase (LDH), acetic acid reacts with adenosine 5′-triphosphate disodium salt trihydrate (ATP) under the catalysis of AK to generate adenosine 5′-diphosphate disodium salt trihydrate (ADP). In the presence of PK, ADP further reacts with phosphoenol pyruvate monopotassium salt (PEP) to generate pyruvate. Then, in the presence of LDH, pyruvate is reduced to lactate by β-nicotinamide adenine dinucleotide reduced disodium salt hydrate (NADH). At the same time, the reduction of NADH will cause a reduction in oxidation current. Thus, a linear relationship between acetic acid concentration and current can be constructed based on the different current values ​​obtained under different acetic acid concentrations. Attached Figure Description

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0030] Figure 1 This is a cyclic voltammogram showing the preparation process of the screen-printed electrode modified with reduced graphene oxide and polymethylene blue in Example 1 of this invention.

[0031] Figure 2 This is a scanning electrode morphology image of the screen-printed electrode modified with reduced graphene oxide and polymethylene blue prepared in Example 1 of the present invention.

[0032] Figure 3 This is a cyclic voltammogram of the bioelectrochemical sensor in Example 1 of the present invention under different acetic acid concentrations.

[0033] Figure 4 This is a linear regression curve of the bioelectrochemical sensor used to detect acetic acid in Example 1 of the present invention. Detailed Implementation

[0034] The present invention will be further described below with reference to the accompanying drawings and specific preferred embodiments, but this does not limit the scope of protection of the present invention.

[0035] Example 1

[0036] A method for detecting acetic acid concentration in an anaerobic fermentation system using a bioelectrochemical sensor, specifically utilizing a bioelectrochemical sensor to detect acetic acid in the anaerobic fermentation broth, includes the following steps:

[0037] (1) Insert the working electrode into the PBS buffer solution and add potassium phosphoenolpyruvate (PEP), adenosine 5′-triphosphate disodium salt trihydrate (ATP), and β-nicotinamide adenine dinucleotide reduced disodium salt hydrate (NADH) to make the concentrations of PEP, ATP, and NADH in the PBS buffer solution 2mM, 5mM, and 10mM, respectively. Connect the electrode to the electrochemical workstation through the lead wire to obtain the bioelectrochemical sensor.

[0038] In step (1), the PBS buffer solution is a 100mM phosphate buffer (KH2PO4, K2HPO4) with pH 7.2 containing 15mM magnesium chloride and 100mM potassium nitrate.

[0039] (2) Turn on the bioelectrochemical sensor and scan in the range of -0.5V to +0.5V using cyclic voltammetry (CV). Add the anaerobic fermentation broth to be tested and scan, and collect the current of the anaerobic fermentation broth at +0.2V.

[0040] (3) Input the current of the anaerobic fermentation broth to be tested into the standard linear regression equation of acetic acid concentration and current, and calculate the concentration of acetic acid in the anaerobic fermentation broth. The results are shown in Table 1.

[0041] In this embodiment, the bioelectrochemical sensor used includes a working electrode, which is a screen-printed electrode. The working surface of the screen-printed electrode is modified with reduced graphene oxide, and polymethylene blue is deposited on the reduced graphene oxide. Acetate kinase, pyruvate kinase and lactate dehydrogenase are connected to the working surface of the screen-printed electrode through a cross-linking agent.

[0042] In this embodiment, the crosslinking agent is polyethylene glycol diglycidyl ether.

[0043] In this embodiment, the method for preparing the working electrode includes the following steps:

[0044] S1. Graphene oxide (GO) powder was dispersed in ultrapure water and sonicated to obtain a graphene oxide solution; 5 μL of a solution with a concentration of 0.3 mg / mL was then added. -1A graphene oxide solution was dropped onto the working surface of a screen-printed electrode and dried at room temperature to obtain a graphene oxide-modified screen-printed electrode, denoted as the GO / SPE electrode.

[0045] S2. Immerse the graphene oxide-modified screen-printed electrode in 5 mL of water. -1 The polymerization reaction was carried out in a methylene blue (MB) solution. During the polymerization, the potential window was -1V to +1.2V, and the scan rate was 10mV / s. -1 The scanning circle number was 15 circles. After the reaction was completed, it was rinsed with ultrapure water to obtain a screen-printed electrode modified with reduced graphene oxide and polymethylene blue, which was denoted as PMB / RGO modified SPE electrode.

[0046] Figure 1 This is a cyclic voltammogram showing the preparation process of the screen-printed electrode modified with reduced graphene oxide and polymethylene blue in Example 1 of this invention. Figure 1 It can be seen that from the 1st cycle to the 15th cycle, the positions of the oxidation peak and the reduction peak shift to higher values, which indicates that the RGO / PMB polymer is obtained by the simultaneous oxidation and reduction of MB and GO.

[0047] Figure 2 This is a scanning electrode morphology image of the screen-printed electrode modified with reduced graphene oxide and polymethylene blue, prepared in Example 1 of this invention. Figure 2 It is evident that a polymer film is present, confirming that the PMB / RGO film was polymerized via an electrochemical method.

[0048] S3. Acetylkinase, pyruvate kinase, and lactate dehydrogenase are mixed with a cross-linking agent according to an enzyme activity ratio of 1:1.5:2 and added to the surface of a screen-printed electrode modified with reduced graphene oxide and polymethylene blue. The mixture is dried at 4°C. Acetylkinase, pyruvate kinase, and lactate dehydrogenase are loaded onto the screen-printed electrode modified with reduced graphene oxide and polymethylene blue to obtain the working electrode.

[0049] In this embodiment, the standard linear regression equation used for acetic acid concentration and current is:

[0050] y = 38.381 × x + 23.9167;

[0051] Where y is the current value of the anaerobic fermentation broth (nA), x is the acetic acid concentration in the anaerobic fermentation broth (mM), and R 2 The value is 0.99578, the linear range is 5mM to 17.5mM, and the detection limit is 2.5mM.

[0052] In this embodiment, the method for constructing the standard linear regression equation between acetic acid concentration and current is as follows:

[0053] Acetic acid standard solutions of different concentrations were added to the bioelectrochemical sensor for scanning, and the results are as follows: Figure 3 As shown.

[0054] Figure 3 This is a cyclic voltammogram of the bioelectrochemical sensor in Example 1 of the present invention under different acetic acid concentrations. Figure 3 It is known that the bioelectrochemical sensor used in this invention has a very large current intensity when used to detect acetic acid, which is more conducive to the detection of higher concentrations of acetic acid.

[0055] Figure 4 This is a linear regression curve of the bioelectrochemical sensor used to detect acetic acid in Example 1 of the present invention.

[0056] In this embodiment, the concentration of acetic acid in the anaerobic fermentation broth to be tested was determined by gas chromatography, and the results are shown in Table 1.

[0057] As shown in Table 1, in this invention, the bioelectrochemical sensor has high accuracy in detecting the acetic acid concentration in anaerobic fermentation broth, whether the concentration is low or high.

[0058] Table 1 Comparison of detection results of the method of the present invention and gas chromatography.

[0059]

[0060] The results above show that the bioelectrochemical sensor used in the method of this invention, under the combined action of reduced graphene oxide and polymethylene blue, can significantly improve the conductivity of the electrode and load more functional enzymes, thereby enabling the sensor to have a faster response speed, a larger current intensity, and advantages such as high sensitivity, wide linear range, and low detection limit. When used to detect the acetic acid concentration in anaerobic fermentation systems, it has advantages such as simple operation, low sample consumption, low price, high precision, fast detection speed, and relatively simple sample pretreatment. It can realize the rapid detection of acetic acid concentration in fermentation systems and can timely and accurately obtain the acetic acid concentration at different stages of anaerobic fermentation, thus providing effective data for anaerobic fermentation analysis, which is of great significance for monitoring the process stability of anaerobic fermentation.

[0061] The above embodiments are merely preferred embodiments of the present invention, and the scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A method for detecting the concentration of acetic acid in an anaerobic fermentation system using a bioelectrochemical sensor, characterized in that, The method is for detecting acetic acid in anaerobic fermentation liquor by using a bioelectrochemical sensor; the bioelectrochemical sensor comprises a working electrode; the working electrode is a screen-printed electrode; the working surface of the screen-printed electrode is modified with reduced graphene oxide, and the reduced graphene oxide is deposited with polymethylene blue; the working surface of the screen-printed electrode is connected with acetic acid kinase, pyruvate kinase and lactate dehydrogenase through a crosslinking agent.

2. The method of claim 1, wherein, The crosslinking agent is polyethylene glycol diglycidyl ether.

3. The method of claim 2, wherein, The preparation method of the working electrode comprises the following steps: S1, dropping a graphene oxide solution onto the working surface of a screen-printed electrode, and drying to obtain a graphene oxide modified screen-printed electrode; S2, immersing the graphene oxide modified screen-printed electrode in a methylene blue solution to perform a polymerization reaction, and obtaining a reduced graphene oxide and polymethylene blue modified screen-printed electrode; S3, mixing acetic acid kinase, pyruvate kinase and lactate dehydrogenase with a crosslinking agent, and adding to the surface of the reduced graphene oxide and polymethylene blue modified screen-printed electrode, and drying, so that the acetic acid kinase, pyruvate kinase and lactate dehydrogenase are loaded on the surface of the screen-printed electrode modified with reduced graphene oxide and polymethylene blue, and a working electrode is obtained.

4. The method of claim 3, wherein, In step S1, the graphene oxide solution is prepared by dispersing graphene oxide into water; the concentration of the graphene oxide solution is 0.1 mg mL -1 ~ 0.5 mg mL -1 .

5. The method of claim 4, wherein, In step S2, the methylene blue solution is prepared by dispersing methylene blue into water; the concentration of the methylene blue solution is 5 mM; the potential window during the polymerization reaction is -1 V to +1.2 V, the scanning rate is 10 mV s -1 ~ 50 mV s -1 and the scanning number of circles is 10 to 20.

6. The method of claim 5, wherein, In step S2, the enzyme activity ratio of the acetic acid kinase, pyruvate kinase and lactate dehydrogenase is 1:1-2:2-3; and the drying is performed at a temperature of 4℃.

7. The method according to any one of claims 1 to 6, characterized in that, When the bioelectrochemical sensor is used to detect acetic acid in anaerobic fermentation liquor, the following steps are included: (1) constructing a bioelectrochemical sensor; (2) adding the anaerobic fermentation liquor to be detected into the bioelectrochemical sensor to perform scanning, and collecting the current of the anaerobic fermentation liquor to be detected; (3) inputting the current of the anaerobic fermentation liquor to be detected into a standard linear regression equation of acetic acid concentration and current, and calculating the concentration of acetic acid in the anaerobic fermentation liquor.

8. The method of claim 7, wherein, In step (3), the standard linear regression equation of acetic acid concentration and current is: y = 38.381 * x + 23.9167; wherein y is the current value (nA) of the anaerobic fermentation broth, x is the acetic acid concentration (mM) in the anaerobic fermentation broth, R 2 is 0.99578, the linear range is 5 mM-17.5 mM, and the detection limit is 2.5 mM.

9. The method of claim 8, wherein, In step (2), the cyclic voltammetry is used to scan the anaerobic fermentation liquor to be detected; and the potential in the scanning process is-0.5V-+0.5V.

10. The method of claim 9, wherein, In step (1), the construction method of the bioelectrochemical sensor comprises the following steps: inserting the working electrode into a PBS buffer solution, adding phosphoenolpyruvate monopotassium salt, adenosine 5'-triphosphate disodium salt trihydrate and beta-nicotinamide adenine dinucleotide reduced disodium salt hydrate, connecting an electrochemical workstation, and obtaining a bioelectrochemical sensor.

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