KR102579378B1 - Method for preparing electron transfer material using biochar produced from microalgae sludge - Google Patents

Abstract

The present invention relates to a method of producing biochar from microalgae sludge, a method of producing an electron transfer material using the produced biochar, and an enzyme fuel cell and biosensor using the produced electron transfer material.
According to the present invention, a new method of using discarded microalgae sludge as a biological resource is provided by utilizing microalgae, producing biochar from discarded microalgae sludge, and using it as an electron transport material.
In addition, the composite of biochar, cobalt hydroxide, and chitosan prepared above is deposited uniformly on the surface of the electrode base, and the enzyme for the oxidation reaction or the enzyme for the reduction reaction is immobilized, resulting in high uniformity and stability and improved enzyme activity. An electrode is provided, and the power amount and sensor sensitivity can be significantly improved by applying the electrode to an enzyme fuel cell or glucose biosensor.

Description

Method for preparing electron transfer material using biochar produced from microalgae sludge}

The present invention relates to a method of producing biochar from microalgae sludge, a method of producing an electron transfer material using the produced biochar, and an enzyme fuel cell and biosensor using the produced electron transfer material.

Microalgae are very small among algae and are autotrophic and unicellular organisms that float freely in water and produce nutrients through photosynthesis. Previously, microalgae were considered unnecessary organisms that simply affected the occurrence of red and green tides, but recently, through their characteristics of producing oxygen through photosynthesis and retaining various substances and nutrients, they have been used to produce bioenergy, future food, and raw materials for cosmetics. As it has become known that it has superior capabilities compared to existing raw materials in various fields, such as reducing greenhouse gases, much research is being conducted to utilize microalgae.

Meanwhile, enzymatic fuel cells have recently attracted the attention of many researchers. Enzyme fuel cells use enzymes as catalysts and are manufactured using materials that are harmless to the human body so that they can operate under conditions such as room temperature, normal pressure, and naturally occurring pH. Their small size makes them suitable for medical and biofuel sensors (biosensors). ), it has many applications in real life, such as batteries for small electronic products.

In such enzyme fuel cells, it is of utmost importance to increase the stability and activity of enzymes and to form an optimized electron transfer environment. Therefore, various studies on electron transporters are being conducted. However, enzyme fuel cells have problems related to the stability of enzymes, such as short lifespan, low current density, and low power density, so continuous research is required to solve these problems.

The present invention was devised to solve the above-mentioned problems, and in the present invention, biochar is produced from discarded microalgae sludge using microalgae, a method of producing an electron transport material using the biochar, and a method of producing an electron transport material using the biochar. The purpose is to provide an enzyme fuel cell and biosensor with high uniformity and stability and improved enzyme activity by including an electron transfer material.

In order to solve the above problems, the present invention

It provides a method for producing biochar including the step of producing biochar by carbonizing microalgae sludge.

According to the present invention, the carbonization can be performed in a nitrogen gas atmosphere and at a temperature of 600-1000° C. for 1-3 hours.

In addition, the present invention includes the steps of reacting CoCl ₂ ·H ₂ O in the solution in which the prepared biochar is dispersed, and then reacting by adding NH ₄ OH to the reaction solution to produce a biochar/cobalt hydroxide composite; and reacting the biochar/cobalt hydroxide composite with a chitosan solution to produce a biochar/cobalt hydroxide/chitosan composite.

According to the present invention, the chitosan solution may include acetic acid as a solvent.

In addition, the present invention relates to an electrode substrate; The electron transport material prepared according to claim 3 deposited on the surface of the electrode substrate; and an electrode containing an enzyme for an oxidation reaction or an enzyme for a reduction reaction immobilized on the surface of the electron transport material.

In addition, the present invention relates to an electrode substrate; The electron transport material prepared according to claim 3 deposited on the surface of the electrode substrate; and an electrode containing an enzyme for oxidation reaction immobilized on the surface of the electron transport material.

According to the present invention, a new method of using discarded microalgae sludge as a biological resource is provided by utilizing microalgae, producing biochar from discarded microalgae sludge, and using it as an electron transport material.

In addition, the composite of biochar, cobalt hydroxide, and chitosan prepared above is deposited uniformly on the surface of the electrode base, and the enzyme for the oxidation reaction or the enzyme for the reduction reaction is immobilized, resulting in high uniformity and stability and improved enzyme activity. An electrode is provided, and the power amount and sensor sensitivity can be significantly improved by applying the electrode to an enzyme fuel cell or glucose biosensor.

Figure 1 shows the results of measuring the mass balance of Chlorella pyrenoidosa algae used in the examples of the present invention.
Figure 2 (a) is an SEM image showing the surface of Chlorella pyrenoidosa algae used in an example of the present invention before the acid extraction process, and (b) is the surface of the algal sludge obtained through the acid extraction process. (c) and (d) are SEM and HRTEM images showing the surface of biochar produced by carbonizing algae sludge.
Figures 3 (a) and (b) are SEM images and HRTEM images showing the surface of the biochar/cobalt hydroxide composite, and (c) is the SEM image showing the surface of the biochar/cobalt hydroxide/chitosan composite and EDX analysis results. It represents.
Figure 4 is a graph showing the results of measuring the cyclic voltage current by applying the biochar/cobalt hydroxide/chitosan composite to the enzyme fuel cell system.
Figure 5 is a graph showing the results of measuring power density by applying a biochar/cobalt hydroxide/chitosan composite to an enzyme fuel cell system.
Figure 6 is a graph showing the results of measuring power density by applying a graphite oxide/cobalt hydroxide/chitosan composite to an enzyme fuel cell system.
Figure 7 is a graph showing the results of measuring sensor sensitivity by applying a biochar/cobalt hydroxide/chitosan composite to a glucose biosensor.

Hereinafter, the present invention will be described in more detail.

The present invention provides a method for producing biochar including the step of carbonizing microalgae sludge to produce biochar.

At this time, the microalgae may be Chlorella or Spirulina, and preferably Chlorella pyrenoidosa .

The microalgae sludge may be obtained by performing an acid extraction process on a powder prepared by drying and pulverizing microalgae.

Additionally, in order to improve the conversion efficiency of the microalgae sludge into biochar, the carbonization is preferably performed for 1-3 hours in a nitrogen gas atmosphere and at a temperature of 600-1000°C.

As can be seen in the examples below, the biochar produced from the microalgae sludge prepared according to the present invention has various surface characteristics and layer structures, and in particular has similar characteristics to graphite oxide, such as a large surface area. It can replace graphite oxide, which is conventionally used as a carbon material for electric energy conversion/storage systems.

In addition, the present invention includes the steps of reacting CoCl ₂ ·H ₂ O in the solution in which the prepared biochar is dispersed, and then reacting by adding NH ₄ OH to the reaction solution to produce a biochar/cobalt hydroxide composite; and reacting the biochar/cobalt hydroxide composite with a chitosan solution to produce a biochar/cobalt hydroxide/chitosan composite.

Cobalt hydroxide is an electrochemical redox active material, and because it has a large layered structure, it is widely used as a material for alkaline batteries, fuel cells, secondary batteries, and supercapacitors.

Therefore, the biochar/cobalt hydroxide composite serves as an electron carrier (mediator) that has the effect of increasing electrochemical properties by combining biochar and cobalt hydroxide.

In addition, the biochar/cobalt hydroxide composite has chitosan uniformly fixed to the surface of the biochar/cobalt hydroxide composite through reaction with the chitosan solution and has high stability, so when applied to an electrode for an enzyme fuel cell, Not only does the stability and activity of the enzyme increase, but it can also improve the power density and current density of the enzyme fuel cell.

Additionally, the present invention provides an enzyme fuel cell and a glucose biosensor containing the above electron transport material.

Specifically, the enzyme fuel cell includes an electrode base; The prepared electron transport material deposited on the surface of the electrode substrate; And it may include an electrode containing an enzyme for an oxidation reaction or an enzyme for a reduction reaction immobilized on the surface of the electron transport material,

The glucose biosensor includes an electrode base; The prepared electron transport material deposited on the surface of the electrode substrate; And it may include an electrode containing an enzyme for oxidation reaction immobilized on the surface of the electron transport material.

At this time, various metal materials and carbon materials such as gold, silver, platinum, copper, aluminum, carbon nanotubes, and graphene may be used as the electrode substrate.

In addition, the deposition of the electron transfer material is, for example, immersed in a solution in which the biochar/cobalt hydroxide/chitosan composite (electron transfer material) is dissolved, and electricity is applied so that the electrode base has a negative charge. This can be performed as a process of depositing the biochar/cobalt hydroxide/chitosan composite on the surface of the electrode substrate.

In addition, the immobilization of the enzyme for the oxidation reaction or the enzyme for the reduction reaction can be performed, for example, using a crosslinking compound of the enzyme for the oxidation reaction or the enzyme for the reduction reaction and the biochar/cobalt hydroxide/chitosan composite.

At this time, EDC and NHS can be used as the cross-linking compounds. More specifically, a solution is prepared by mixing an enzyme for an oxidation reaction or an enzyme for a reduction reaction with the cross-linking compounds EDC and NHS, and the biochar/cobalt hydroxide is added thereto. /It can be performed by immersing the electrode base on which the chitosan composite is deposited on the surface so that the enzyme for the oxidation reaction or the enzyme for the reduction reaction is immobilized on the surface of the biochar/cobalt hydroxide/chitosan composite through EDC and NHS.

The EDC and NHS combine an enzyme for an oxidation reaction or an enzyme for a reduction reaction with the biochar/cobalt hydroxide/chitosan composite by covalent bonding.

As for the method of fixing the enzyme for the oxidation reaction or the enzyme for the reduction reaction to the surface of the graphite oxide/cobalt hydroxide/chitosan composite, methods known in the field other than the method using covalent bonding may be used without limitation.

At this time, the enzymes for the oxidation reaction and the enzymes for the reduction reaction may be those known in the field. For example, when glucose is used as a substrate, the enzymes for the oxidation reaction include glucose oxidase and glucose dehydrogenase. Glucose dehydrogenase, cellobiose dehydrogenase, dehydrogenase, etc. can be used, and enzymes for reduction reactions include laccase and horseradish peroxidase. , bilirubin oxidase, etc. can be used.

Hereinafter, the present invention will be described in more detail, including preferred embodiments. However, these examples are for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited thereto.

Example 1. Birds sludge powder manufacturing

As a microalgae, Chlorella pyrenoidosa ( Chlorella pyrenoidosa ) powder prepared by drying WUDI LV QI BIOENGINEERING Co., Ltd. Purchased from (China). Algal sludge was prepared through an acid extraction process for 1 kg of the microalgae powder. Specifically, the microalgae powder was set to a solid/liquid ratio of 100 g/L and an acid concentration of 2% in a 20 mL vial, and heat-treated in a sterilizer at 121°C for 15 minutes. Algae sludge powder (900 g) was prepared by drying the sludge, excluding useful substances from the hydrolyzed supernatant through centrifugation, and powdering it.

Example 2. Birds from sludge biochar manufacturing

The algal sludge powder prepared in Example 1 was placed in an alumina sample dish and carbonized at 800 degrees for 2 hours while injecting nitrogen gas (300 ml/min) to prepare biochar (270 g).

Example 3. biochar /Manufacture of cobalt hydroxide/chitosan composite (electron transport material)

(One) biochar /Manufacture of cobalt hydroxide composite

0.1 g of biochar prepared through Example 2 was dispersed in 10 ml of distilled water. Then, 2M CoCl ₂ ·H ₂ O was dissolved in 10 ml of distilled water. These two solutions were mixed and stirred for 24 hours to allow cobalt ions to be adsorbed onto the biochar. After the reaction, NH ₄ OH was added to adjust the pH to 9.0 and stirred for 24 hours so that the cobalt ions adsorbed on the biochar became cobalt hydroxide. The biochar/cobalt composite thus formed was washed with distilled water and dried in a vacuum oven at 60°C for 2 hours.

(2) biochar /Manufacture of cobalt hydroxide/chitosan composite

0.1 g of chitosan was dissolved in a mixed solution of 100 ml of 25×TAE (Tris-acetate-EDTA) buffer and 3% acetic acid (100°C), and then filtered through a nylon filter. Next, a biochar/cobalt hydroxide/chitosan composite was prepared by mixing 3 g of the biochar/cobalt hydroxide composite and 0.1 wt% of the chitosan solution (relative to the total weight of the mixture) and coating the biochar/cobalt hydroxide composite with the chitosan solution.

Example 4. Enzyme fuel cell manufacturing

The Au anode and Au cathode of the power supply were immersed in the biochar/cobalt hydroxide/chitosan composite solution, and 50 V was applied to deposit the biochar/cobalt hydroxide/chitosan composite on the Au electrode for 2 minutes. The biochar/cobalt hydroxide/chitosan composite is deposited on the electrically negative Au electrode because the dissolved chitosan has a positive charge.

Next, in order to manufacture an anode or cathode on which an enzyme for an oxidation or reduction reaction is immobilized, glucose, an enzyme for an oxidation reaction, is added to 0.1M phosphate buffer (pH 7.0). oxidase or laccase, an enzyme for reduction reaction, at 01 mg/ml, 05 mg/ml, 1 mg/ml, 2 mg/ml, 3 mg/ml, and 4 mg/ml, respectively, were mixed, and 0.12M EDC and 0.14M NHS were mixed together. After mixing, the Au electrode on which the biochar/cobalt hydroxide/chitosan composite was deposited was immersed in the mixed solution at 4°C for 8 hours. At this time, the enzyme is immobilized on the biochar/cobalt hydroxide/chitosan composite by the EDC/NHS reaction. This reaction occurs by covalent bonding between the carboxyl group in the enzyme and the amine group in chitosan. After allowing the immobilization to proceed, it was washed with 0.1M phosphate buffer and tertiary distilled water.

Next, the electrode on which glucose oxidase was immobilized on the biochar/cobalt hydroxide/chitosan composite was used as an anode, and the electrode on which Lacase reductase was immobilized was used as a cathode, and were connected to each other using 0.05M phosphoric acid. A basic enzyme fuel cell without an ion membrane was manufactured using an electrolyte containing 1% glucose in a buffer (pH 7.0).

Example 5. Glucose biosensor manufacturing

A basic glucose biosensor was manufactured using the electrode on which glucose oxidase was immobilized on the biochar/cobalt hydroxide/chitosan composite prepared through the method disclosed in Example 4 as an anode.

Test example 1. Mass balance measurement of microalgae

Figure 1 shows the results of measuring the mass balance of Chlorella pyrenoidosa algae used in the examples of the present invention. As shown in Figure 1, when the acid extraction process was performed on 1 kg of microalgae powder in Example 1, 4.6 g of oil and 63.5 g of hydrolyzate were produced, and the remaining sludge excluding useful substances such as oil and hydrolyzate was measured as 900 g. It was confirmed that when the carbonization process as in Example 2 was performed on the microalgae sludge, 270 g of biochar was produced.

Test example 2. Microalgae, microalgae sludge and of biochar surface analysis

Figure 2 (a) is an SEM image showing the surface before pretreatment of Chlorella pyrenoidosa algae used in the examples of the present invention, and it was confirmed that the microalgae had a spherical shape before the acid extraction process.

Figure 2 (b) is an SEM image showing the surface of microalgae sludge obtained through an acid extraction process. It was confirmed that the cell wall skeleton was decomposed by acid extraction and the surface structure of the spherical cells was deformed.

Figure 2(c) is an SEM image showing the surface of biochar produced by carbonizing algae sludge. The surface of biochar undergoes dehydration and decomposition of carbonic acid through the carbonization process, causing oxygen to form O ₂ , CO, and CO ₂ It was confirmed that the surface was modified as the volatile matter was almost removed and only the fixed carbon remained.

Figure 2 (d) is an SEM and HRTEM image showing the surface of biochar produced by carbonizing algae sludge. It was confirmed that the biochar particles appear to be surrounded by a thin film and the thin plates form a layered structure, making it graphite. It was confirmed that it had similar characteristics to oxide.

Finally, as a result of performing EDX analysis on biochar (not shown), the composition of biochar was found to be C: 96.7% and O: 0.8%.

Test example 3. biochar /Cobalt hydroxide composite and biochar /Surface analysis of cobalt hydroxide/chitosan composite

Figures 3 (a) and (b) are SEM images and HRTEM images showing the surface of the biochar/cobalt hydroxide composite, and (c) is the SEM image showing the surface of the biochar/cobalt hydroxide/chitosan composite and EDX analysis results. It represents.

Through Figure 3, it was confirmed that cobalt hydroxide particles were adsorbed on the surface of biochar, and it was confirmed that chitosan chains were coated on the surface of biochar/cobalt hydroxide. In addition, it was confirmed that 5.4% of N was generated in EDX analysis, which confirmed that an amine group was formed and that the synthesis of the biochar/cobalt hydroxide/chitosan composite as an electron transport material was successful.

Test example 4. Evaluation of voltage-current and power production of enzyme fuel cells, measurement of sensitivity of glucose biosensor

For the enzyme fuel cell manufactured in Example 4, voltage-current and power production were evaluated by measuring cyclic voltammetry and power density using 0.1M glucose as a substrate. Specifically, it was connected to a VersaSTAT 3 device (AMETEK, Princeton Applied Research, USA) and voltage-current and power density were measured at 25°C.

Figure 4 is a graph showing the results of measuring the cyclic voltage current by applying the biochar/cobalt hydroxide/chitosan composite to the enzyme fuel cell system. Specifically, cyclic voltammetry was measured in the voltage range of -0.6 to 0.6 V at a scanning rate of 100 mV/s at room temperature, and it was confirmed that the oxidation reaction peak increased significantly.

Figure 5 is a graph showing the results of measuring power density by applying a biochar/cobalt hydroxide/chitosan composite to an enzyme fuel cell system. Specifically, Figure 5 shows the relationship between the current density and voltage of the EFC along with the respective power densities, and as a result of measurement, it was confirmed that the current density was 9.7 mA/cm ² and the power density was 3.1 mW/cm ² .

For comparison, the graphite oxide/cobalt hydroxide/chitosan composite was applied to an enzyme fuel cell system and the power density was measured and shown in Figure 6. Through this, when the conventional graphite oxide/cobalt hydroxide/chitosan composite is applied to the enzyme fuel cell system, the power output is about 3 times lower than when the biochar/cobalt hydroxide/chitosan composite according to the present invention is applied to the enzyme fuel cell system, which is 1 mW/cm ² It was confirmed that the electron transfer ability of the biochar/cobalt hydroxide/chitosan composite according to the present invention was very high.

Next, the glucose biosensor manufactured according to Example 5 was connected to an ersaSTAT 3 device (AMETEK, Princeton Applied Research, USA) and sensitivity was measured by adding 2mM glucose at 25°C. The results are shown in FIG. 7. Figure 7 shows a general current-concentration response when 2mM of glucose is continuously added. The current density for electrochemical oxidation of glucose increased with the glucose concentration, and the sensitivity was 0.488 mA/mM cm ² , excellent It was confirmed that it shows sensitivity.

Claims (6)

delete delete delete delete electrode substrate;
An electron transport material deposited on the surface of the electrode substrate; and
It includes an electrode containing an enzyme for an oxidation reaction or an enzyme for a reduction reaction immobilized on the surface of the electron transport material,
The electron transport material is reacted by adding CoCl ₂ ·H ₂ O to a solution in which biochar is dispersed, and then reacting by adding NH ₄ OH to the reaction solution to produce a biochar/cobalt hydroxide composite; And reacting the biochar/cobalt hydroxide composite with a chitosan solution to produce a biochar/cobalt hydroxide/chitosan composite, wherein the biochar is prepared by mixing microalgae sludge in a nitrogen gas atmosphere and at a temperature of 600-1000°C. -An enzyme fuel cell manufactured by carbonization for 3 hours. electrode substrate;
An electron transport material deposited on the surface of the electrode substrate; and
It includes an electrode containing an enzyme for oxidation reaction immobilized on the surface of the electron transport material,
The electron transport material is reacted by adding CoCl ₂ ·H ₂ O to a solution in which biochar is dispersed, and then reacting by adding NH ₄ OH to the reaction solution to produce a biochar/cobalt hydroxide composite; And reacting the biochar/cobalt hydroxide composite with a chitosan solution to produce a biochar/cobalt hydroxide/chitosan composite, wherein the biochar is prepared by mixing microalgae sludge in a nitrogen gas atmosphere and at a temperature of 600-1000°C. -A glucose biosensor manufactured by carbonization for 3 hours.