Method for producing ammonia and mixing

Description

1 / 14 METHOD FOR PRODUCING AMMONIA AND MIXING TECHNICAL FIELD

[001] The present invention relates to a method for producing ammonia and a mixture. BACKGROUND

[002] Current industrial methods of ammonia production are mainly those in which ammonia is synthesized from hydrogen and nitrogen by means of a catalyst that contains iron as the main component (Haber-Bosch process).

[003] In the Haber-Bosch process, hydrogen and nitrogen are induced to react under conditions of high temperature and high pressure (400-500°C and 100-300 atm). Therefore, a method to which biological nitrogen fixation is applied has attracted attention as a method in which ammonia is produced under milder conditions. For example, Patent Document 1 describes a process in which ammonia is produced using recombinant microorganisms that exhibit nitrogen-fixing activity.

[004] Patent Document 1: Japanese Laid-Open Patent Application No. 2021-145675 SUMMARY OF THE INVENTION Problem to be Solved by the Invention

[005] Biological nitrogen fixation is a process in which an enzyme (nitrogenase) present in a certain type of microorganism converts nitrogen molecules from the air into a nitrogen compound, such as ammonia. Since biological nitrogen fixation occurs at normal temperatures and pressures, it is considered a promising method of ammonia production with a lower environmental impact than conventional chemical production methods. However, improved production efficiency is needed to achieve practical use of biological nitrogen fixation.

[006] In view of the foregoing, the purpose of this disclosure is to provide a method for producing ammonia that Petition 870250088707, dated 09 / 30 / 2025, page 13 / 34 2 / 14 utilizes biological nitrogen fixation and exhibits excellent ammonia production efficiency, and a mixture that exhibits excellent ammonia production efficiency. Ways to Solve the Problem

[007] This disclosure includes the following modalities.

[008] <1> Ammonia production methods including: To prepare a modified cyanobacterium in which the rate of heterocysts is increased in the cells that constitute a nitrogen-fixing cyanobacterium; and to apply light, in the presence of nitrogen, to a mixture including the modified cyanobacterium, a photocatalyst, and water.

[009] <2> Ammonia production method, according to <1> , in which the mixture also includes glycerol.

[010] <3> Ammonia production method, according to <1> or <2> , in which the mixture also includes an electron mediator.

[011] <4> Ammonia production method, according to any of <1> the <3> , in which the mixture also includes a nitrogen metabolism inhibitor.

[012] <5> Ammonia production method, according to any of <1> the <4> , in which the photocatalyst includes titanium oxide.

[013] <6> Ammonia production method, according to any of <1> the <5> , in which the preparation of the modified cyanobacteria is carried out by cultivating a nitrogen-fixing cyanobacterium in a culture that does not include a nitrogen source.

[014] <7> A mixture that is used for the ammonia production method according to any of <1> the <6> , the mixture including; a modified cyanobacterium, in which the heterocyst rate is increased in the cells that constitute a nitrogen-fixing cyanobacterium; Petition 870250088707, dated 09 / 30 / 2025, page 14 / 34 3 / 14 a photocatalyst; and water.

[015] <8> Mixture including; a modified cyanobacterium, in which the heterocyst rate is increased in the cells that constitute a nitrogen-fixing cyanobacterium; a photocatalyst; and water.

[016] <9> Mixture, according to <8> including glycerol.

[017] <10> Mixture, according to <8> or <9> , including an electron mediator.

[018] <11> A mixture, according to anyone of <8> a < 10>, also including a nitrogen metabolism inhibitor.

[019] <12> A mixture, according to anyone of <8> a < 11>, also including titanium oxide.

[020] <13> A mixture, according to anyone of <8> a < 12>, which is for the production of ammonia.

[021] <14> Use the mixture according to any of the <8> the <12> , which is for the production of ammonia. EFFECTS OF THE INVENTION

[022] According to the present disclosure, a method of ammonia production that utilizes biological nitrogen fixation and exhibits excellent ammonia production efficiency, and a mixture that exhibits excellent ammonia production efficiency are provided. BRIEF DESCRIPTION OF THE INVENTION

[023] FIG. 1 is a graph that illustrates the metal content in nitrogen-fixing cyanobacteria. MODALITIES FOR IMPLEMENTING THE INVENTION

[024] In this disclosure, any numerical interval described using the expression from ... to ... represents an interval in which the numerical values ​​described before and after Petition 870250088707, dated 09 / 30 / 2025, page 15 / 34 4 / 14 of a are included in the interval as a minimum and a maximum value, respectively.

[025] In a numerical range described in a gradual manner in this disclosure, an upper limit value or a lower limit value described in one numerical range may be replaced by an upper limit value or a lower limit value in another numerical range described in a gradual manner. Furthermore, in a numerical range described in this disclosure, the upper limit value or the lower limit value in the numerical range may be replaced by a value shown in the Examples.

[026] In this disclosure, a component may include several types of substances corresponding to the component. In a case where a respective component in a composition includes several types of substances, the content of the respective component refers to the total content of the various types of substances present in the composition, unless otherwise specified.

[027] The ammonia production method of the present disclosure includes: To prepare a modified cyanobacterium in which the rate of heterocysts is increased in the cells that constitute a nitrogen-fixing cyanobacterium; and to apply light, in the presence of nitrogen, to a mixture including the modified cyanobacterium, a photocatalyst, and water.

[028] The ammonia production method of the present disclosure utilizes biological nitrogen fixation. Specifically, the method produces ammonia through the nitrogen-fixing activity of a nitrogen-fixing cyanobacterium.

[029] In this disclosure, nitrogen-fixing cyanobacteria refers to a cyanobacterium that has the ability to fix nitrogen. Petition 870250088707, dated 09 / 30 / 2025, page 16 / 34 5 / 14

[030] The general description of a process for producing ammonia by the method of this disclosure is as follows.

[031] When light is applied to a mixture that includes a modified cyanobacterium, a photocatalyst, and water, the light-absorbing photocatalyst decomposes water molecules into hydrogen and oxygen. The electron (e-) generated by the decomposition of water molecules is used for the nitrogen fixation reaction (N2 + 8H++ 8e- ^ 2NH3 + H2) caused by the nitrogenase included in the heterocysts. The ammonia produced by the nitrogen fixation reaction of the nitrogenase is protected by glycerol against decomposition resulting from the metabolic reaction and released from the cells.

[032] In the method of the present disclosure, the efficiency of ammonia production is improved by the following means.

[033] (1) A modified cyanobacterium, in which the rate of heterocysts in the cells is increased, is used as the nitrogen-fixing cyanobacterium.

[034] Nitrogen-fixing cyanobacteria (hereinafter referred to simply as cyanobacteria) are composed of cells called heterocysts, which contain nitrogenase and serve for nitrogen fixation, and vegetative cells that do not contain nitrogenase and serve for photosynthesis. In the modified cyanobacteria used in the method of the present disclosure, a potential nitrogen-fixing capacity (i.e., the total amount of nitrogenase present in heterocysts) is enhanced compared to a cyanobacterium that has not been modified. Furthermore, in the modified cyanobacteria used in the method of the present disclosure, a substantial nitrogen-fixing capacity (i.e., the work rate of the nitrogenase present in the heterocysts) is improved by increasing the opportunity for heterocysts to receive electrons generated by the decomposition of water molecules caused by a photocatalyst, compared to a case where the amount Petition 870250088707, dated 09 / 30 / 2025, page 17 / 34 6 / 14 of the cyanobacteria used for ammonia production is simply increased.

[035] (2) A photocatalyst is used in conjunction with cyanobacteria.

[036] The rate of ammonia production via the nitrogen fixation reaction of nitrogenase is extremely low, as a large amount of ATP (adenosine triphosphate) is consumed as a metabolic reaction in cells. In the method of the present disclosure, nitrogenase in heterocysts is supplied with electrons generated by the decomposition of water molecules caused by a photocatalyst. Therefore, the nitrogen fixation reaction of nitrogenase is promoted without being restricted by the amount of ATP to be supplied. (Nitrogen-fixing cyanobacteria)

[037] The type of nitrogen-fixing cyanobacteria used in the method of this disclosure is not restricted. For example, filamentous cyanobacteria such as Anabaena sp. PCC7120, Anabaena variabilis ATCC29413 and Nostoc punctiforme ATCC29133 can be used.

[038] The nitrogen-fixing cyanobacteria used in the method of the present disclosure may be of a single type or a combination of two or more types.

[039] The method for obtaining a modified cyanobacterium by increasing the rate of heterocysts in the cells that constitute a nitrogen-fixing cyanobacterium is not particularly limited. For example, the modified cyanobacterium can be obtained by cultivating a nitrogen-fixing cyanobacterium under conditions in which heterocyst differentiation is induced. For example, since heterocyst formation is caused by nitrogen deficiency, a modified cyanobacterium can be obtained by cultivating a nitrogen-fixing cyanobacterium in a culture medium that does not include a nitrogen source, such as an Allen & Arnon medium (AA medium) and a Petition 870250088707, dated 09 / 30 / 2025, page 18 / 34 7 / 14 BG11 medium (a culture medium obtained by removing sodium nitrate from a BG11 medium).

[040] Alternatively, a modified cyanobacterium can be obtained by a technique such as genetic recombination.

[041] It is possible to determine whether the heterocyst rate in the cells that constitute the modified cyanobacteria has increased or not due to a treatment to obtain the modified cyanobacteria, for example, based on the metal content in the cyanobacteria. Specifically, when the Mo and Fe content in the cyanobacteria after treatment is higher than that of the cyanobacteria before treatment, it can be determined that the amount of Fe-Mo type nitrogenase present in the heterocysts has increased. Thus, it can be determined that a modified cyanobacterium is obtained, in which the heterocyst rate in the cells is increased. (Photocatalyst)

[042] The photocatalyst used in the method of the present disclosure is not particularly limited, provided it is a substance that has the ability to absorb light and decompose water into hydrogen and oxygen.

[043] Specific examples of photocatalysts include titanium oxide (TiO2), gallium nitride (GaN) and StTiO3. From the point of view of catalytic activity and availability, titanium oxide is preferable as the photocatalyst.

[044] From the point of view of catalytic activity, the photocatalyst is preferably in powder form. The particle size of the photocatalyst in powder form should be as small as possible, preferably 300 nm or less, more preferably 200 nm or less.

[045] The specific surface area of ​​the photocatalyst is preferably 50 m2 / g or more. The specific surface area of ​​the photocatalyst is a value measured by a BET method using nitrogen gas. Petition 870250088707, dated 09 / 30 / 2025, p. 19 / 34 8 / 14

[046] The photocatalyst used in the method of the present disclosure may be of a single type or a combination of two or more types. (Glycerol)

[047] The mixture used in the method of the present disclosure also preferably includes glycerol. As demonstrated in the Examples described later, when the mixture includes glycerol, the amount of hydrogen generation is increased, as well as the amount of ammonia generation. From the above results, it is considered that glycerol has the function of increasing the activity of hydrogenase, which is an enzyme included in the modified cyanobacteria.

[048] Glycerol also acts as a sacrificial reagent for the photocatalyst (a substance that donates an electron to the photocatalyst). (Electron Mediator)

[049] The mixture used in this disclosure preferably includes an electron mediator. When the mixture includes an electron mediator, the electrons generated by the action of the photocatalyst are effectively supplied to the heterocysts of the modified cyanobacteria. As a result, the nitrogen fixation reaction caused within the heterocysts is promoted.

[050] Specific examples of electron mediators include viologen compounds, such as methyl viologen dichloride and benzyl viologen dichloride, 2-amino-2-hydroxymethyl-1,3propanediol, triethanolamine (TEOA), bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis-Tris) and triethylamine (TEA).

[051] The electron mediator used in the method of the present disclosure may be of a single type or a combination of two or more types.

[052] From the point of view of the photocatalyst's ability to transport electrons to heterocysts, the compounds Petition 870250088707, dated 09 / 30 / 2025, page 20 / 34 9 / 14 of viologen are preferable as electron mediators. (Nitrogen Metabolism Inhibitor)

[053] The mixture used in the method of the present disclosure also preferably includes a nitrogen metabolism inhibitor. The nitrogen metabolism inhibitor inhibits the activity of glutamine synthetase (an enzyme that generates glutamine from glutamic acid and ammonia) by acting in opposition to glutamic acid, thereby suppressing ammonia consumption by the modified cyanobacteria. As a result, the efficiency of ammonia production is increased.

[054] Specific examples of nitrogen metabolism inhibitors include sulfoximine methionine, bialafos and glufosinate.

[055] The nitrogen metabolism inhibitor used in the method of the present disclosure may be of a single type or a combination of two or more types.

[056] As needed, the mixture may include a component other than the modified cyanobacteria, photocatalyst, glycerol, electron mediator, and nitrogen metabolism inhibitor. Examples of the other component include a pH adjuster, a buffer, and a phosphate, such as nicotinamide adenine dinucleotide phosphate (NADPH).

[057] The content of modified cyanobacteria in the mixture can be selected in a range of 0.05 g / mL to 0.25 g / mL, for example.

[058] The photocatalyst content in the mixture can be selected in a range of 0.1 mg / mL to 2 mg / mL, for example.

[059] The glycerol content in the mixture can be selected in a range of 10 mmol / L to 100 mmol / L, for example.

[060] The content of the electron mediator in the mixture can be selected in a range of 1 mmol / L to 20 mmol / L, for example.

[061] The nitrogen metabolism inhibitor content in the mixture can be selected in a range from 0.1 μmol / L to 1 Petition 870250088707, dated 09 / 30 / 2025, p. 21 / 34 10 / 14 mmol / L, preferably from 1 μmol / L to 100 μmol / L.

[062] The ratio of modified cyanobacteria content to photocatalyst (modified cyanobacteria : photocatalyst) is preferably selected in a range of 100:0.1 to 100:25, more preferably in a range of 100:1 to 100:10.

[063] The light applied to the mixture is not particularly limited, provided it includes light with a wavelength at which the photocatalyst is excited. The amount of light to be applied is not particularly limited and can be determined depending on the amount of mixture, the amount of components included in the mixture, or something similar. The temperature of the mixture to which the light is applied can be selected within a range of 20°C to 40°C, for example. EXAMPLES

[064] The modalities of this disclosure are described in more detail below based on the Examples. However, the present disclosure is not limited to the Examples. (Preparation of Cyanobacteria)

[065] A cyanobacterium for the ammonia production test was prepared by cultivating a nitrogen-fixing cyanobacterium in an AA medium or a BG11 medium under light irradiation of 5,000 Lux (68 μmol / m2 / s), while the medium was stirred at 26°C and 140 rpm. Anabaena variabilis ATCC29413 (acquired from the American Type Culture Collection) was used as the nitrogen-fixing cyanobacterium.

[066] The amounts of Fe, Ni, and Mo in the cyanobacteria obtained were measured by ICP-AES atomic emission spectrometry. The results are shown in FIG. 1. In the cyanobacteria obtained in BG11 medium, the amounts of Fe and Ni indicate that Fe-Ni hydrogenase is dominant and FeMo nitrogenase is not increased. On the other hand, in the cyanobacteria obtained in AA medium, the significantly high Mo content indicates that Fe-Mo nitrogenase is increased. Petition 870250088707, dated 09 / 30 / 2025, page 22 / 34 11 / 14

[067] Based on the results above, it was determined that the cyanobacteria obtained in medium AA (Cyano AA) was a modified cyanobacteria and the cyanobacteria obtained in medium BG11 (Cyano BG11) was not a modified cyanobacteria. (Ammonia Production Test 1)

[068] Modified cyanobacteria (Cyano AA), comparative cyanobacteria (Cyano BG11), glycerol (Gly), titanium oxide (T1O2, particle size: 140 nm, specific surface area: 53 m2 / g) and methyl viologen (MV2+) in the quantities indicated in Table 1 were mixed with water, thus preparing the mixtures (100 mL, pH 7.5).

[069] Each of the mixtures was placed in a reactor made of quartz, and light was applied to the mixture under conditions of 30°C and 400 mW / cm2 (light source: xenon lamp) while nitrogen gas was supplied to the mixture.

[070] The amount of hydrogen produced and the amount of nitrogen consumed during light irradiation were measured using a TCD-type gas chromatograph connected to an online system. The amount of ammonia produced was measured using an ion chromatograph (Dionex, USA). The results 72 hours after the start of light irradiation are presented in Table 1. TABLE 1 Cyan AA Cyan BG11 Gly TiO2 mv2+ H2 Production N2 Consumption nh4+ Production [g] [g] [mM] [mg] [mM] [pmol] [mmol] [pmol] Comparative Example 1 5 - 100 - 17.4 0.078 21.44 Comparative Example 2 - - 100 250 -9.5 0.071 12.61 Example 1 5 - 100 250 -63.1 0.131 32.47 Example 2 5 - 250 10 25.9 0.135 35.67 Example 3 5 - 100 250 10 130.9 0.427 69.23 Comparative Example 3 - 5 100 250 10 141.8 0.051 20.42

[071] As shown in Table 1, Example 1, in which the light Petition 870250088707, dated 09 / 30 / 2025, page 23 / 34 The 12 / 14 method applied to the mixture including the modified cyanobacteria, T1O2, and glycerol in the presence of nitrogen, exhibits a greater amount of ammonia produced compared to Comparative Example 1, in which the mixture includes the modified cyanobacteria and glycerol but does not include TiO2, and to Comparative Example 2, in which the mixture includes TiO2 and glycerol but does not include the modified cyanobacteria.

[072] The reason why the amount of ammonia produced in Example 1 is greater than in Comparative Example 1 is considered to be that the inherent ability of the cyanobacteria to produce ammonia is enhanced by TiO2.

[073] It is considered that the reason why the amount of ammonia produced in Example 1 is greater than in Comparative Example 2 is that the amount of ammonia produced due to the photocatalytic action of TiO2 is increased by the modified cyanobacteria.

[074] Example 1, in which the mixture includes glycerol in addition to the modified cyanobacteria and TiO2, shows a greater amount of ammonia and hydrogen produced in total than Example 2, in which the mixture does not include glycerol. It is considered that the reason for this is that the hydrogenase activity in the cyanobacteria is intensified and the amount of hydrogen produced is increased by the glycerol, which acts as a sacrificial reagent that promotes the photocatalytic action of TiO2. From the results above, it is verified that glycerol contributes to an increase in the amounts of ammonia and hydrogen produced.

[075] Example 3, in which the mixture includes MV2+ in addition to the modified cyanobacteria, TiO2 and glycerol, shows a greater amount of ammonia produced than Example 1, in which the mixture does not include MV2+. Based on the previous results, it was found that MV2+ contributes to an increase in the amount of ammonia produced.

[076] Example 3, in which the mixture includes cyanobacteria Petition 870250088707, dated 09 / 30 / 2025, page 24 / 34 The modified cyanobacterium 13 / 14, in which the heterocyst rate was increased, exhibits a greater amount of ammonia produced than Comparative Example 3, in which the mixture includes the comparative cyanobacterium in which the heterocyst rate was not increased. From the results above, it is verified that the modification of the cyanobacterium contributes to an increase in the amount of ammonia produced. (Ammonia Production Test 2)

[077] Modified cyanobacteria (Cyano AA), glycerol (Gly), titanium oxide (TiO2, particle size: 140 nm, specific surface area: 53 m2 / g) and methyl viologen (MV2+) in the amounts indicated in Table 2 were mixed with water, thus preparing the mixtures (100 mL, pH 7.5).

[078] The mixtures were subjected to the ammonia production test in the same manner as ammonia production test 1. The results 24 hours after the start of light irradiation are presented in Table 2. TABLE 2 Cyan AA Gly TiO2 MV2+ H2 Production N2 Consumption NH4+ Production [g] [mM] [mg] [mM] [pmol] [mmol] [pmol] Example 4 1 50 250 10 59.51 0.173 21.89 Example 5 3 50 250 10 98.67 0.269 44.27 Example 6 6 50 250 10 203.15 0.512 65.08

[079] As shown in Table 2, it can be observed that the amount of ammonia produced tends to increase as the amount of modified cyanobacteria in the mixture is increased. The previous results indicate that the modified cyanobacteria contributes to an increase in the amount of ammonia produced. (Ammonia Production Test 3)

[080] Modified cyanobacteria (Cyano AA), glycerol (Gly), titanium oxide (T1O2, particle size: 140 nm, specific surface area: 53 m2 / g), methyl viologen (MV2+) Petition 870250088707, dated 09 / 30 / 2025, page 25 / 34 14 / 14 and methionine sulfoximine (MSX) in the amounts indicated in Table 2 were mixed with water, thus preparing the mixtures (100 mL, pH 7.5).

[081] The mixtures were subjected to the ammonia production test in the same manner as ammonia production test 1. The results at 72 hours from the start of light irradiation are presented in Table 3. TABLE 3 Cyan AA Gly TiO2 MV2+ MSX H2 Production N2 Consumption NH4+ Production [g] [mM] [mg] [mM] [μM] ^mol ] [mmol] ^mol ] Example 2 5 100 250 10 _ 130.9 0.427 69.23 Example 7 5 100 250 10 10 194.2 0.481 87.06

[082] Example 7, in which the mixture includes MSX in addition to the modified cyanobacteria, TiO2, glycerol, and MV2+, shows a greater amount of ammonia produced than Example 2, in which the mixture does not include MSX. It is thought that the reason for this is that a decrease in ammonia due to cyanobacteria metabolism is inhibited by MSX, which acts as a nitrogen metabolism inhibitor. From the results above, it is verified that the nitrogen metabolism inhibitor contributes to an increase in the amount of ammonia produced.

[083] The disclosure of Japanese Patent Application No. 2023062401 is incorporated herein by reference in its entirety. All publications, patent applications and technical standards mentioned in this descriptive report are incorporated herein by reference to the same extent as if each individual publication, patent application or technical standard were specifically and individually indicated for incorporation by reference. Petition 870250088707, dated 09 / 30 / 2025, pp. 26 / 34

Claims

1 / 2 CLAIMS 1. A method for producing ammonia, characterized in that it comprises: preparing a modified cyanobacterium in which the rate of heterocysts is increased in the cells that constitute a nitrogen-fixing cyanobacterium; and applying light, in the presence of nitrogen, to a mixture including the modified cyanobacterium, a photocatalyst, and water.

2. A method for producing ammonia, according to claim 1, characterized in that the mixture also includes glycerol.

3. A method for producing ammonia, according to claim 1 or 2, characterized in that the mixture further includes an electron mediator.

4. A method for producing ammonia, according to any one of claims 1 to 3, characterized in that the mixture further includes a nitrogen metabolism inhibitor.

5. A method for producing ammonia, according to any one of claims 1 to 4, characterized in that the photocatalyst includes titanium oxide.

6. A method for producing ammonia, according to any one of claims 1 to 5, characterized in that the preparation of the modified cyanobacteria is carried out by cultivating a nitrogen-fixing cyanobacterium in a culture that does not include a nitrogen source.

7. A mixture used for the method of producing ammonia as defined in any one of claims 1 to 6, the mixture characterized in that it comprises: a modified cyanobacterium, in which the rate of heterocysts is increased in the cells constituting a nitrogen-fixing cyanobacterium; a photocatalyst; and water. Petition 870250088707, dated 09 / 30 / 2025, p. 31 / 34 2 / 2 8. A mixture characterized by comprising: a modified cyanobacterium, in which the rate of heterocysts is increased in the cells that constitute a nitrogen-fixing cyanobacterium; a photocatalyst; and water.

9. Mixture according to claim 8, characterized in that it further comprises glycerol.

10. Mixture according to claim 8 or 9, characterized in that it further comprises an electron mediator.

11. Mixture, according to any one of claims 8 to 10, characterized in that it further comprises a nitrogen metabolism inhibitor.

12. Mixture, according to any one of claims 8 to 11, characterized in that it further comprises titanium oxide.

13. Mixture, according to any one of claims 8 to 12, characterized in that it is for the production of ammonia.

14. Use of the mixture as defined in any one of claims 8 to 12, characterized by the fact that it is for the production of ammonia. Petition 870250088707, dated 09 / 30 / 2025, pp. 32 / 34

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