JP5709125B2 - Nitrous oxide decomposition equipment - Google Patents

Description

  The present invention relates to a nitrous oxide decomposition apparatus.

Nitrous oxide (N ₂ O) has a greenhouse effect about 300 times higher than that of carbon dioxide, and is subject to emission regulations in the Kyoto Protocol. For this reason, from the viewpoint of preventing global warming, it is required to efficiently remove / decompose nitrous oxide discharged from factories, agricultural land, and the like.

  As a method for decomposing nitrous oxide, a method using a metal catalyst has been disclosed so far (for example, Patent Document 1).

JP 2007-152263 A

  The method using a metal catalyst requires a reaction at a high temperature in order to decompose nitrous oxide, and requires a large amount of energy to increase the temperature. For example, in the method disclosed in Patent Document 1, the processing temperature when decomposing nitrous oxide is 450 ° C.

  Then, an object of this invention is to provide the nitrous oxide decomposition | disassembly apparatus which can decompose | disassemble nitrous oxide energy-saving at normal temperature.

  The present invention provides a nitrous oxide decomposing apparatus comprising a cathode composed of an enzyme electrode having nitrous oxide reductase and an anode connected to the cathode.

  The nitrous oxide decomposition apparatus of the present invention can decompose nitrous oxide using electrons generated at the anode by the action of nitrous oxide reductase at the cathode. Since the nitrous oxide decomposition apparatus of the present invention is based on such an action, it can decompose nitrous oxide at room temperature. This eliminates the need for high-temperature heating conventionally required, and is advantageous in terms of energy. In addition, since nitrous oxide reductase is used, the substrate specificity is high and only nitrous oxide can be specifically decomposed. In addition, the nitrous oxide decomposition apparatus of the present invention is self-supporting and does not require an external power source for applying a voltage.

  It is preferable that the anode includes a biological catalyst that can generate electrons from an organic substance. When the anode contains such a biological catalyst, organic substances can be used as an electron source, so that nitrous oxide can be decomposed at a lower cost.

  The biological catalyst can be at least one selected from the group consisting of enzymes, microorganisms, and biofilms.

  The nitrous oxide reductase is preferably one that forms a complex with cytochrome C. Since nitrous oxide reductase forms a complex with cytochrome C, electrons can be directly transferred from the cathode to nitrous oxide reductase via cytochrome C, so that oxygen or the like exists. In some cases, nitrous oxide can be decomposed more efficiently without electrons being taken away by oxygen or the like.

  The nitrous oxide reductase is more preferably one having cytochrome C in the domain, and the nitrous oxide reductase can be derived from Wolinella succinogenes.

  The present invention is also a nitrous oxide decomposition method using the nitrous oxide decomposition apparatus, wherein the cathode and a sample containing nitrous oxide are brought into contact with each other, and the electrons generated in the anode are used to Provided is a nitrous oxide decomposition method for decomposing the nitrous oxide by the action of nitrous oxide reductase.

  The nitrous oxide decomposition method of the present invention is capable of decomposing nitrous oxide at room temperature, which eliminates the need for conventional high-temperature heating and is advantageous in terms of energy. In addition, since nitrous oxide reductase is used, the substrate specificity is high and only nitrous oxide can be specifically decomposed.

  According to the present invention, there is provided a nitrous oxide decomposition apparatus capable of decomposing nitrous oxide in an energy-saving manner at room temperature. According to the nitrous oxide decomposition apparatus of the present invention, nitrous oxide can be decomposed at room temperature. This eliminates the need for conventional high-temperature heating, is advantageous in terms of energy, and has the advantage that the amount of carbon dioxide generated is small. In addition, since nitrous oxide reductase is used, the substrate specificity is high and only nitrous oxide can be specifically decomposed. In addition, no external power source is required to move electrons to the cathode.

  Moreover, since the reducing power (energy) required for nitrous oxide decomposition can be obtained from organic substances, organic wastes and the like can be used as reducing power. Therefore, nitrous oxide originating from agricultural land or the like can be efficiently reduced, which is useful as a global warming countermeasure technique.

It is a schematic diagram of the reaction tank which concerns on one Embodiment of the nitrous oxide decomposition | disassembly apparatus of this invention. It is a schematic diagram of the reaction tank which concerns on other embodiment of the nitrous oxide decomposition | disassembly apparatus of this invention. 2 is a graph showing nitrous oxide decomposition in Example 2. 3 is a graph showing nitrous oxide decomposition in Example 3.

  Hereinafter, preferred embodiments of the present invention will be described.

  The nitrous oxide decomposing apparatus of the present invention comprises a cathode composed of an enzyme electrode having at least nitrous oxide reductase, and an anode connected to the cathode.

A reaction tank 100 according to an embodiment of the nitrous oxide (N ₂ O) decomposition apparatus of the present invention is shown in FIG. The reaction tank 100 shown in FIG. 1 has an enzyme electrode (N ₂ OR electrode) on which a nitrous oxide reductase 1 is immobilized as a cathode 11, and is a biological catalyst that can generate electrons from organic matter (decomposition of organic matter). Means) An electrode having 2 is provided as the anode 12. The sample solution 51 on the cathode 11 side and the sample solution 52 on the anode 12 side are partitioned by an ion exchange membrane 31 and connected by a salt bridge 32.

  At the anode 12, electrons generated when the organic substance contained in the sample solution 52 is decomposed by the organic substance decomposing means 2 are supplied to the nitrous oxide reductase 1 of the cathode 11 through the lead 21, and the supplied electrons are supplied. , Nitrous oxide contained in the sample solution 51 is selectively decomposed by the nitrous oxide reductase 1. In the nitrous oxide decomposition apparatus of the present invention, electrons can move through the leads 21 without requiring voltage supply from an external power source.

Nitrous oxide reductase 1 is an enzyme that catalyzes a reaction represented by the following formula (1).
N ₂ O + 2H ⁺ + 2e ⁻ → N ₂ + H ₂ O (1)
Decomposition of nitrous oxide by nitrous oxide reductase 1 requires electrons (reducing power) as is apparent from the above formula (1). Therefore, even if nitrous oxide reductase 1 and nitrous oxide are simply brought into contact with each other, decomposition of nitrous oxide does not occur. In the nitrous oxide decomposition apparatus according to the present embodiment, by supplying electrons necessary for the decomposition as electrons generated along with the decomposition of the organic matter by the organic matter decomposing means 2, nitrous oxide can be saved at room temperature in an energy saving manner. It becomes possible to disassemble.

  The nitrous oxide reductase 1 is not particularly limited as long as it is an enzyme that can catalyze the reaction represented by the above formula (1). Further, from the viewpoint of efficiently performing the nitrous oxide decomposition reaction at the cathode 11, the nitrous oxide reductase 1 is preferably one that forms a complex with cytochrome C. As a result, electrons are directly supplied from the cathode 11 to the nitrous oxide reductase 1 via the cytochrome C. Therefore, even when an electron acceptor such as dissolved oxygen is present in the sample solution 51, it becomes possible to supply electrons to the nitrous oxide reductase 1 without depriving these electron acceptors, Nitrous oxide can be decomposed more efficiently.

  More preferably, nitrous oxide reductase 1 has cytochrome C in the domain. “Having in a domain” means a state of binding to nitrous oxide reductase 1 through a hydrogen bond or a covalent bond. As an example of such a nitrous oxide reductase 1, specifically, a nitrous oxide reductase derived from Worinella succinogenes can be preferably used.

  The nitrous oxide reductase 1 can be a purified and commercially available nitrous oxide reductase, or can be extracted / purified from a microorganism expressing the nitrous oxide reductase. A method for extracting / purifying nitrous oxide reductase from a microorganism expressing nitrous oxide reductase can be performed according to a conventional protein purification method known to those skilled in the art. Hereinafter, as an example, W.W. A method for extracting / purifying nitrous oxide reductase derived from Succinogenes species is described.

  W. succinogenes is a kind of rumen bacterium and can be obtained from a plurality of public institutions. For example, it can be easily obtained from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) in Germany or ATCC (American Type Culture Collection) in the United States.

  W. The nitrous oxide reductase derived from succinogenes can be used, for example, in W. succinogenes were inoculated and cultured at 37 ° C. under anaerobic conditions with aeration of nitrous oxide throughout the culture. It can be purified from succinogenes cells or culture broth using enzyme activity as an index.

  W. Examples of the liquid medium used for culturing succinogenes include a medium containing yeast extract, sodium formate, sodium citrate, copper sulfate, thioglycolic acid, and having a pH adjusted to near neutral. In addition, a nutrient medium obtained by adding a nutrient medium component, trypticase peptone, vitamin K1, hemin and the like to the above liquid medium is added to W. Succinogenes may be inoculated and cultured at 37 ° C. under anaerobic conditions.

  W. As a specific purification method of succinogenes-derived nitrous oxide reductase, for example, cultured W. The supernatant (crude enzyme solution containing nitrous oxide reductase) obtained by crushing the cells of succinogenes with a French press or an ultrasonic crusher and centrifuging them is subjected to ion exchange chromatography, hydroxyapatite chromatography, etc. It is possible to purify as a single enzyme by fractionation using the purification means.

The enzyme electrode (N ₂ OR electrode) on which the nitrous oxide reductase 1 used as the cathode 11 is immobilized can be produced by mixing the nitrous oxide reductase 1 with an electrode material such as carbon (carbon paste). As an electrode material, platinum or gold can be used in addition to carbon. Alternatively, platinum-supported carbon may be used.

  As a method for immobilizing nitrous oxide reductase 1 on an electrode material, for example, a method using a crosslinking reagent, a method of encapsulating in a polymer matrix, a method of coating with a dialysis membrane, a photocrosslinking polymer or a redox polymer is used. Can be used in combination, and these may be used in combination. More specifically, there is a method of blocking glutaraldehyde by immobilizing the above nitrous oxide reductase 1 on a carbon electrode using glutaraldehyde and then treating with a reagent having an amine group.

Specifically, for example, Lion Paste W-311N (Lion Corporation): 5% Nafion (DuPont Corporation): 5 mM potassium phosphate buffer (pH 6.8): 46 mU / μl 1 nitrous oxide reductase solution 5 μl was added dropwise to a polished glassy carbon (GC) electrode (φ3 mm) after being mixed at a volume ratio of 1: 2: 6, dried at 4 ° C. for 2 hours, and then a 25% glutaraldehyde solution vapor was applied. The N ₂ OR electrode can be obtained by crosslinking for 30 minutes and immersing in 1 mM Tris-HCl (pH 7.0) for 20 minutes and further in 100 mM potassium phosphate buffer (pH 7.0) for 30 minutes.

  The anode 12 includes an organic matter decomposing means 2 composed of a biological catalyst that can generate electrons from organic matter. The anode 12 decomposes the organic matter by the catalytic activity of the organic matter decomposing means 2 and leads the electrons generated along with the decomposition. What is necessary is just to supply to the cathode 11 through 21.

  The organic substance decomposing means 2 is preferably at least one selected from the group consisting of enzymes, microorganisms and biofilms. Specific examples of such enzymes include enzymes that catalyze redox reactions of organic substances. In addition, such a microorganism may be any microorganism that has an enzyme that catalyzes a redox reaction of an organic substance, and preferably has an enzyme that catalyzes a redox reaction of an organic substance on the surface of the microorganism. For example, it can be a microorganism that can be used for the anode of a microbial fuel cell. Moreover, the biofilm formed with such microorganisms may be sufficient.

  The organic substance is not particularly limited as long as it is decomposed by an enzyme, a microorganism, or the like to generate electrons, and can be appropriately set according to the kind of the enzyme, the microorganism, or the like. Moreover, according to the kind of said organic substance which can be utilized, you may set suitably the kind, such as an enzyme and microorganisms.

  Specific examples of organic substances include sugars such as glucose, lower fatty acids, organic acids, alcohols, amino acids, and the like. These organic substances may be a combination of a plurality of types. In addition, organic wastes present in the environment such as a waste disposal site, a sewage treatment plant, and farmland can be used as organic matter.

In the present embodiment, the reaction vessel 100 may include an electron mediator in the sample solution 51. An electronic mediator can exchange electrons. Since the electron mediator promotes the movement of electrons from the cathode 11 to the nitrous oxide reductase 1, nitrous oxide can be decomposed more efficiently. The electron mediator may be included in the N ₂ OR electrode.

  As the electron mediator, for example, benzyl viologen, methyl viologen, potassium ferricyanide, phenazine methosulfate (PMS), 1-methoxyphenazine methosulfate (mPMS), ferrocene derivative, osmium derivative and the like can be used.

  The sample solution 51 is not particularly limited as long as it does not inhibit the enzyme activity of the nitrous oxide reductase 1. Similarly, the sample solution 52 is not particularly limited as long as it does not inhibit the biological catalyst activity contained in the organic matter decomposition means 2. For example, as the sample liquids 51 and 52, waste water containing organic waste or the like can be used.

The reaction tank 110 which concerns on other embodiment for implementing the nitrous oxide decomposition | disassembly apparatus of this invention was shown in FIG. The reaction tank 110 shown in FIG. 2 has an enzyme electrode (N ₂ OR electrode) on which the nitrous oxide reductase 1 is immobilized as the cathode 11, and an electrode provided with the organic matter decomposing means 2 as the anode 12. . The cathode 11 and the anode 12 are immersed in the sample solution 50.

  At the anode 12, electrons generated when the organic substance contained in the sample solution 50 is decomposed by the organic substance decomposing means 2 are supplied to the nitrous oxide reductase 1 of the cathode 11 through the lead 21, and the supplied electrons are supplied. , Nitrous oxide contained in the sample solution 50 is selectively decomposed by the nitrous oxide reductase 1.

  The sample solution 50 is not particularly limited as long as it does not inhibit the enzyme activity of the nitrous oxide reductase 1 and the catalytic activity of the organic matter decomposition means 2. For example, as the sample solution 50, waste water containing organic waste or the like can be used.

  The nitrous oxide decomposing apparatus of the present invention can decompose nitrous oxide at room temperature and can decompose nitrous oxide as long as organic substances are present, so there is no need to add energy such as heat from the outside. Cost is advantageous.

  Since the nitrous oxide decomposition apparatus of the present invention does not require an external power source, it should be suitably used for nitrous oxide decomposition in the environment (for example, agricultural land, disposal site, waste disposal site, sewage treatment plant, etc.). Can do.

  For example, by piercing a cathode and an anode made of an enzyme electrode having nitrous oxide reductase into soil such as farmland, electrons are generated from organic matter in the soil by the catalytic activity of the anode, and the electrons are used to generate sub-electrons at the cathode. Nitrogen oxide can be decomposed. In addition, when decomposing nitrous oxide contained in aeration gas at a sewage treatment plant or the like, the aeration gas is generated using electrons generated from organic substances in the sewage by placing the cathode in the gas phase and immersing the anode in the sewage. It can also decompose nitrous oxide.

  Hereinafter, the present invention will be described in more detail with reference to examples.

Example 1 Production of Electrode Immobilized with Nitrous Oxide Reductase (N ₂ OR Electrode) Nitrous oxide reductase was obtained from Wolinella succinogenes ATCC 29543 by the method described in International Publication WO2009 / 031652. Further, by the method described in International Publication WO2009 / 031652 pamphlet, to give nitrous oxide reductase enzyme immobilized electrode _(N 2 OR electrodes).

Decomposition of nitrous oxide (Example _{2) N} 2 OR electrodes (mediator added)
An N ₂ OR electrode was used as the cathode. On the other hand, on the anode, an electrode (GDH electrode) with immobilized glucose dehydrogenase prepared in the same manner as the N ₂ OR electrode except that glucose dehydrogenase (FADGDH) was used instead of nitrous oxide reductase was used. Using. These electrodes were installed in a reaction tank filled with 100 mM phosphate buffer (pH 7.0), and the cathode side sample solution and the anode side sample solution were partitioned by an ion exchange membrane. Glucose was added to the anode side sample solution to 10 mM, and mPMS was added to the cathode side sample solution to 2 mM as an electron mediator. The cathode and anode were connected to a potentiostat (see FIG. 1 for the configuration). Argon gas or nitrous oxide gas was bubbled into the cathode side sample solution, and the current value at that time was measured with a potentiostat.

  The results are shown in FIG. There was a clear difference between the power generated when argon gas was bubbled into the sample solution on the cathode side and the power generated when bubbling nitrous oxide (FIG. 3). From this result, it was confirmed that nitrous oxide was reduced, that is, decomposed by the action of nitrous oxide reductase at the cathode using electrons generated by oxidation of glucose at the anode.

(Example 3) Decomposition of nitrous oxide using N ₂ OR electrode (without addition of mediator)
The N ₂ OR electrode was used as the cathode and the GDH electrode was used as the anode. These electrodes were placed in a reaction tank filled with 100 mM phosphate buffer (pH 7.0). Unlike Example 2, the sample solution on the cathode side and the sample solution on the anode side were not separated by an ion exchange membrane, and were made into a single tank type reaction vessel. Also, no electron mediator was added. Glucose was added to the sample solution to 10 mM, and the cathode and anode were connected to a potentiostat (see FIG. 2 for the configuration). The sample solution was bubbled with argon gas or nitrous oxide gas, and the current value at that time was measured with a potentiostat.

The results are shown in FIG. When argon gas is bubbled into the sample solution and when nitrous oxide is bubbled, there is a difference in the power generated, and the power generated when bubbling nitrous oxide is generated when argon gas is bubbled. It was several times the power (Fig. 4). As a result, it was confirmed that nitrous oxide was selectively reduced, that is, decomposed by the action of nitrous oxide reductase at the cathode using electrons generated by oxidation of glucose at the anode. This result also shows that electrons are transferred directly from the N ₂ OR electrode to nitrous oxide reductase without the addition of an electron mediator.

  From the above examples, it was shown that the nitrous oxide decomposition apparatus of the present invention does not require the supply of voltage from an external power source, and can decompose nitrous oxide using electrons generated at the anode.

  DESCRIPTION OF SYMBOLS 1 ... Nitrous oxide reductase, 2 ... Organic substance decomposition | disassembly means, 11 ... Cathode, 12 ... Anode, 21 ... Lead, 31 ... Ion-exchange membrane, 32 ... Salt bridge, 50, 51, 52 ... Sample solution, 100, 110 ... Reaction tank.

Claims (6)

A cathode composed of an enzyme electrode having nitrous oxide reductase, and an anode connected to the cathode ,
A nitrous oxide decomposing apparatus , wherein the anode includes a biological catalyst capable of generating electrons from organic matter . The nitrous oxide decomposing apparatus according to claim 1, wherein the biological catalyst is at least one selected from the group consisting of an enzyme, a microorganism, and a biofilm. The nitrous oxide decomposing apparatus according to claim 1 or 2 , wherein the nitrous oxide reductase forms a complex with cytochrome C. The nitrous oxide reductase according to any one of claims 1 to 3 , wherein the nitrous oxide reductase has cytochrome C in the domain. The nitrous oxide reductase is derived from Worinera-Sakushinogenesu (Wolinella succinogenes), nitrous oxide decomposition apparatus according to any one of claims 1-4. A nitrous oxide decomposition method using the nitrous oxide decomposition apparatus according to any one of claims 1 to 5 ,
A nitrous oxide decomposition method in which the cathode and a sample containing nitrous oxide are brought into contact, and the nitrous oxide is decomposed by the action of the nitrous oxide reductase using electrons generated in the anode.

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