Method for producing ammonium nitrate, method for producing nitric acid, device for producing ammonium nitrate, and device for producing nitric acid

Abstract

In this method for producing ammonium nitrate, ammonia is supplied to a reaction liquid including water, ammonia-oxidizing bacteria, and nitrite-oxidizing bacteria that include at least bacteria belonging to the family Xanthobacteraceae, the ammonia is converted into nitric acid by the metabolic action of the ammonia-oxidizing bacteria and the nitrite-oxidizing bacteria, ammonium nitrate is further generated by a neutralization reaction of the ammonia and the nitric acid, and the ammonium nitrate is accumulated in the reaction liquid.

Description

Ammonium nitrate manufacturing method, nitric acid manufacturing method, ammonium nitrate manufacturing apparatus, and nitric acid manufacturing apparatus

[0001] The present invention relates to a method for producing ammonium nitrate using ammonia-oxidizing bacteria and nitrite-oxidizing bacteria, a method for producing nitric acid, an apparatus for producing ammonium nitrate, and an apparatus for producing nitric acid. This application claims priority to Japanese Patent Application No. 2024-137849, filed on August 19, 2024, the contents of which are incorporated herein by reference.

[0002] Conventionally, a water purification method using a bacterial composition (consortium) suitable for nitrifying a liquid containing ammonia and nitrite has been proposed (for example, Patent Document 1).

[0003] Patent No. 5107715

[0004] RSC Adv., 2018, 8, 31987-31995Water Res. 2011, 45 (15), 4672-4682.

[0005] Manure from livestock farms is sometimes composted and disposed of. However, the ammonia gas generated during this process is harmful if released into the atmosphere, so treatment facilities must be located in mountain forests and consideration must be given to nearby residents. Therefore, there is a need to recover and effectively utilize the ammonia gas.

[0006] The present invention provides a method for producing industrially useful nitrate nitrogen by dissolving ammonia gas in water and treating it with microorganisms, and an apparatus suitable for such production.

[0007] [1] A method for producing ammonium nitrate, comprising: supplying ammonia to a reaction solution containing water, ammonia-oxidizing bacteria, and nitrite-oxidizing bacteria; converting the ammonia to nitrate through metabolic action of the ammonia-oxidizing bacteria and the nitrite-oxidizing bacteria; generating ammonium nitrate through a neutralization reaction between the ammonia and the nitrate; and accumulating the ammonium nitrate in the reaction solution. In this embodiment, the nitrite-oxidizing bacteria preferably include at least bacteria belonging to the family Xanthobacteraceae. [2] The method for producing ammonium nitrate according to [1], further comprising adding sodium nitrate to the reaction solution before supplying ammonia to the reaction solution, thereby increasing the nitrate ion concentration of the reaction solution to 2000 mg-N / L or more. [3] The method for producing ammonium nitrate according to [1] or [2], wherein the proportion of reads classified as Nitrospiraceae in the reaction solution at the start of ammonium nitrate production, as measured by 16S rRNA amplicon sequencing, is 6% or less of the total number of 16S rRNA amplicon reads in the reaction solution. For example, it is preferable that the above amplicon sequencing is performed on the reaction solution before supplying ammonia to the reaction solution, and the above ratio of reads is used. [4] A method for producing ammonium nitrate according to any one of [1] to [3], wherein the pH of the reaction solution is maintained in the range of 6.0 to 8.0. [5] A method for producing ammonium nitrate according to any one of [1] to [4], wherein an excess amount of ammonia exceeding the metabolic activity is supplied to the reaction solution. [6] A method for producing ammonium nitrate according to any one of [1] to [5], wherein an inorganic alkali is supplied to the reaction solution. [7] A method for producing ammonium nitrate according to any one of [1] to [6], wherein the supply of ammonia is continued until the concentration of nitrate nitrogen accumulated in the reaction solution reaches 5000 mg-N / L or more. [8] A method for producing ammonium nitrate according to any one of [1] to [7], wherein the supply of ammonia is continued until the concentration of ammonium nitrate accumulated in the reaction solution reaches 5000 mg / L or more.[9] A method for producing nitric acid, comprising supplying ammonia to a reaction solution containing water, ammonia-oxidizing bacteria, and nitrite-oxidizing bacteria, converting the ammonia to nitric acid through metabolic action of the ammonia-oxidizing bacteria and nitrite-oxidizing bacteria, and extracting nitric acid from the reaction solution by electrodialysis.

[10] A method for producing nitric acid according to [9], wherein an inorganic alkali is added to the reaction solution to adjust the pH of the reaction solution to 6.0 to 8.0.

[11] A method for producing nitric acid according to [9] or

[10] , which extracts the inorganic alkali together with nitric acid from the reaction solution by electrodialysis and returns the extracted inorganic alkali to the reaction solution.

[12] An apparatus for producing ammonium nitrate, comprising: an ammonia supply unit; a reaction tank for holding a reaction solution containing water, ammonia-oxidizing bacteria, and nitrite-oxidizing bacteria; and a pH controller, wherein the ammonia supply unit supplies ammonia to the reaction tank, and the pH controller controls the amount of ammonia supplied from the ammonia supply unit in accordance with changes in the pH of the reaction solution, so that ammonium nitrate accumulates in the reaction solution.

[13] The apparatus for producing ammonium nitrate according to

[12] , further comprising an inorganic alkali supply unit that supplies inorganic alkali to the reaction tank, and the pH controller is configured to control the amount of inorganic alkali supplied from the inorganic alkali supply unit in accordance with changes in the pH of the reaction solution.

[14] The apparatus for producing ammonium nitrate according to

[12] or

[13] , wherein the pH controller controls the pH of the reaction solution to be 6.0 to 8.0.

[15] An apparatus for producing nitric acid, comprising an ammonia supply unit, a reaction tank that holds a reaction solution containing water, ammonia-oxidizing bacteria, and nitrite-oxidizing bacteria, and an electrodialysis device, wherein the ammonia supply unit supplies ammonia to the reaction tank, and the electrodialysis device extracts nitric acid from the reaction solution.

[16] The apparatus for producing nitric acid according to

[15] , wherein the electrodialysis device extracts ammonium ions together with nitric acid from the reaction solution and returns the extracted ammonium ions to the reaction tank.

[17] The apparatus for producing nitric acid according to

[15] or

[16] , further comprising an inorganic alkali supply unit that supplies an inorganic alkali to the reaction tank, and the electrodialysis device extracts the inorganic alkali together with nitric acid from the reaction solution and returns the extracted inorganic alkali to the reaction tank.

[18] The apparatus for producing nitric acid according to

[17] , further comprising a pH controller that controls the amount of ammonia supplied from the ammonia supply unit, the amount of inorganic alkali supplied from the inorganic alkali supply unit, or the amount of ammonium ion or inorganic alkali returned from the electrodialysis device, depending on a change in the pH of the reaction solution, so that the pH of the reaction solution is 6.0 to 8.0.

[0008] According to the present invention, a method for producing industrially useful nitrate nitrogen by dissolving ammonia gas in water and treating it with microorganisms, and an apparatus suitable for the production can be provided. Furthermore, according to the present invention, biomass such as manure, which has been treated as waste in livestock farming, can be utilized as an effective resource.

[0009] 1 is a schematic diagram showing an example of the configuration of an apparatus for producing ammonium nitrate; 2 is a schematic diagram showing an example of the configuration of an apparatus for producing nitric acid; 3 is a graph showing the change in nitrogen concentration of each form over time during the acclimation process of a nitrifying bacteria consortium; (A) shows the change in nitrogen concentration of each form over time during the ammonia supply stop period, and (B) shows the change in nitrogen concentration of each form over time during the ammonia supply period; 4 is a graph showing the change in pH or nitrogen concentration of each form over time in nitrification reactor #1; (A) shows the change in pH over time; (B) shows the change in NO3 - (C) shows the change over time of NH4 + or NO2 - (D) shows the change over time in free ammonia nitrogen (FAN) or free nitrite nitrogen (FNN). The gray background indicates the period when ammonia supply was stopped and the period when sodium hydroxide solution was supplied to adjust the pH. Graphs showing the change over time in pH or the concentration of each form of nitrogen in nitrification reactor #2. (A) shows the change over time in pH. (B) shows the change over time in NO3 - (C) shows the change over time of NH4 + or NO2-(D) shows the time-dependent changes in free ammonia nitrogen (FAN) or free nitrite nitrogen (FNN). The gray background indicates periods when ammonia supply was suspended and periods when sodium hydroxide solution was supplied for pH adjustment. The figure shows the relative abundance of bacterial families with a maximum relative abundance of 3% or greater in nitrification reactor #1. From the middle to the latter stages of operation, groups whose relative abundance increased with increasing nitrate ion concentration are shown in the upper cluster, and groups whose relative abundance decreased are shown in the lower cluster. In the figure, the cutoff height represents the distance (dissimilarity) threshold used to define clusters by cutting the dendrogram. The figure shows the relative abundance of bacterial families with a maximum relative abundance of 3% or greater in nitrification reactor #2. From the middle to the latter stages of operation, groups whose relative abundance increased with increasing nitrate ion concentration are shown in the upper cluster, and groups whose relative abundance decreased are shown in the lower cluster. The horizontal axis represents the number of days since the start of operation, and the values ​​shown represent abundance. In the figure, the cutoff height represents the distance (dissimilarity) threshold used to define clusters by cutting the dendrogram. The graph shows the change over time in the relative abundance of bacterial genera belonging to major families that may be involved in the nitrification process in nitrification reactor #1. (A) shows the change over time in the relative abundance of bacterial genera belonging to the Nitrosomonadaceae family. The horizontal axis shows the number of days elapsed since the start of operation, and the vertical axis shows the relative abundance. (B) shows the change over time in the relative abundance of bacterial genera belonging to the Nitrospiraceae family. The horizontal axis shows the number of days elapsed since the start of operation, and the vertical axis shows the relative abundance. (C) shows the change over time in the relative abundance of bacterial genera belonging to the Xanthobacteraceae family. The horizontal axis shows the number of days elapsed since the start of operation, and the vertical axis shows the relative abundance. The graph shows the change over time in the relative abundance of bacterial genera belonging to major families that may be involved in the nitrification process in nitrification reactor #2. (A) shows the change over time in the relative abundance of bacterial genera belonging to the Nitrosomonadaceae family. The horizontal axis shows the number of days elapsed since the start of operation, and the vertical axis shows the relative abundance. (B) shows the change over time in the relative abundance of bacterial genera belonging to the Nitrospiraceae family. The horizontal axis shows the number of days since the start of operation, and the vertical axis shows the relative abundance. (C) shows the change over time in the relative abundance of bacterial genera belonging to the Xanthobacteraceae family. The horizontal axis shows the number of days since the start of operation, and the vertical axis shows the relative abundance.The graph shows the change over time in the relative abundance of Feature IDs of bacterial species belonging to the Xanthobacteraceae family. The horizontal axis shows the number of days since operation began, and the vertical axis shows relative abundance. (A) Nitrification reactor #1. (B) Nitrification reactor #2. The graph shows the Feature IDs with the highest relative abundance in nitrification reactors #1 and #2, and the phylogenetic tree of bacteria closely related to them. The graph shows the relative abundance of bacterial species based on full-length 16S rRNA sequencing performed on day 84 of nitrification reactor #2. NO3 in nitrification reactors #3-5. - or NO2 -(A) shows the change over time in nitrification reactor #3. (B) shows the change over time in nitrification reactor #4. (C) shows the change over time in nitrification reactor #5. (A) shows the change over time in nitrification reactor #3. (B) shows the change over time in nitrification reactor #4. (C) shows the change over time in nitrification reactor #5. (A) shows the change over time in the reaction rate constant k for the ammonia oxidation reaction. (B) shows the change over time in the relative abundance of bacterial genera belonging to major families that may be involved in the nitrification process in nitrification reactor #3. (A) shows the change over time in the relative abundance of bacterial genera belonging to the Nitrosomonadaceae family. The horizontal axis shows the number of days since the start of operation, and the vertical axis shows the relative abundance. (B) shows the change over time in the relative abundance of bacterial genera belonging to the Nitrospiraceae family. The horizontal axis shows the number of days since the start of operation, and the vertical axis shows the relative abundance. (C) shows the change over time in the relative abundance of bacterial genera belonging to the family Xanthobacteraceae. The horizontal axis shows the number of days since the start of operation, and the vertical axis shows relative abundance. This figure shows the change over time in the relative abundance of bacterial genera belonging to major families that may be involved in the nitrification process in nitrification reactor #4. (A) shows the change over time in the relative abundance of bacterial genera belonging to the family Nitrosomonadaceae. The horizontal axis shows the number of days since the start of operation, and the vertical axis shows relative abundance. (B) shows the change over time in the relative abundance of bacterial genera belonging to the family Nitrospiraceae. The horizontal axis shows the number of days since the start of operation, and the vertical axis shows relative abundance. (C) shows the change over time in the relative abundance of bacterial genera belonging to the family Xanthobacteraceae. The horizontal axis shows the number of days since the start of operation, and the vertical axis shows relative abundance. This figure shows the change over time in the relative abundance of bacterial genera belonging to major families that may be involved in the nitrification process in nitrification reactor #5. (A) shows the change over time in the relative abundance of bacterial genera belonging to the Nitrosomonadaceae family. The horizontal axis shows the number of days since the start of operation, and the vertical axis shows the relative abundance. (B) shows the change over time in the relative abundance of bacterial genera belonging to the Nitrospiraceae family. The horizontal axis shows the number of days since the start of operation, and the vertical axis shows the relative abundance. (C) shows the change over time in the relative abundance of bacterial genera belonging to the Xanthobacteraceae family. The horizontal axis shows the number of days since the start of operation, and the vertical axis shows the relative abundance.This shows the change over time in the relative abundance of Feature IDs of bacterial species belonging to the Xanthobacteraceae family. The horizontal axis shows the number of days since operation began, and the vertical axis shows the relative abundance. (A) is nitrification reactor #3. (B) is nitrification reactor #4. (C) is nitrification reactor #5. This shows the phylogenetic tree of Feature IDs with high relative abundance in nitrification reactors #3-5, and their closely related bacteria.

[0010] <<Method for Producing Ammonium Nitrate>> A first aspect of the present invention is a method for producing ammonium nitrate by supplying ammonia (NH3) to a reaction solution containing water, ammonia-oxidizing bacteria (AOB), and nitrite-oxidizing bacteria (NOB), converting the ammonia to nitric acid (HNO3) through metabolic action of the AOB and NOB, and then producing ammonium nitrate through a neutralization reaction between the ammonia and the nitric acid, and accumulating the ammonium nitrate in the reaction solution. In the production method of this aspect, it is preferable that the nitrite-oxidizing bacteria include at least bacteria belonging to the family Xanthobacteraceae, from the viewpoint of sufficient accumulation of ammonium nitrate.

[0011] Ammonia oxidizing bacteria is a general term for bacteria that oxidize ammonia to nitrite, and is abbreviated as AOB. The AOB used in this embodiment is not particularly limited as long as it is capable of oxidizing ammonia supplied to the reaction solution to nitrite. One type of AOB may be used, or two or more types of AOB may be used in combination. Specific examples include those classified into the genera Nitrosomonas, Nitrosococcus, Nitrosospira, Nitrosovibrio, and Nitrosolobus. The AOB used in this embodiment may be a commercially available microbial preparation, or may be AOB obtained from soil or sludge from a sewage treatment plant using known microbiological techniques.

[0012] Nitrite oxidizing bacteria is a general term for bacteria that oxidize nitrite to nitrate, and is abbreviated as NOB. The NOB used in this embodiment is not particularly limited as long as it is capable of oxidizing nitrite in the reaction solution to nitrate, and preferably includes at least bacteria belonging to the Xanthobacteraceae family. Furthermore, one type of NOB may be used in addition to the Xanthobacteraceae family, or two or more types of NOB may be used in combination with the Xanthobacteraceae family. Specific examples include those classified into the genera Nitrospira, Nitrobacter, and Nitrococcus. When the NOB used in this embodiment includes Nitrospiraceae, the proportion of reads (i.e., the number of DNA sequence fragments) classified as Nitrospiraceae in the reaction solution at the start of operation (i.e., any time point when ammonium nitrate production is initiated), as measured by 16S rRNA amplicon sequencing, is preferably 6% or less, more preferably 5.7% or less, of the total number of 16S rRNA amplicon reads in the reaction solution. Conventional methods for 16S rRNA amplicon sequencing can be used. The NOB used in this embodiment may be a commercially available microbial preparation or NOB obtained from soil or sludge from a sewage treatment plant using known microbiological techniques. Furthermore, the nitrite concentration of the NOB culture solution can be gradually increased to obtain NOB acclimatized to a high concentration of nitrite. By using this NOB, ammonium nitrate can be accumulated at a high concentration in the reaction solution.

[0013] The main medium of the reaction solution is water, and AOB and NOB may be dispersed in water and allowed to freely diffuse, or may be held in a known microbial support and immersed in water. Examples of microbial supports include polymer hydroxygels such as alginic acid. AOB and NOB held in the microbial support exert a metabolic effect on the reaction solution soaked in the microbial support. The generated nitric acid naturally diffuses out of the microbial support.

[0014] In one embodiment, the reaction solution may be a water dispersion of mud containing AOB and NOB. If it is necessary to increase the activity of NOB and balance it with the high activity of AOB, it is preferable to prepare a reaction solution that has been previously acclimatized with a high concentration of nitrous acid.

[0015] The amount of AOB contained in the reaction solution is, for example, 1 × 10 ６ ~1 x 10 １０ The amount of NOB contained in the reaction solution may be in the range of, for example, 1 × 10 ６ ~1 x 10 １０ The ratio of AOB to NOB contained in the reaction solution, expressed as AOB / NOB, is, for example, in the range of 0.2 to 2.5, or in the range of 0.2 or more but less than 1.0, etc.

[0016] The reaction solution may contain any additives in addition to water, AOB, and NOB. Examples of the additives include inorganic alkalis such as sodium hydroxide for controlling the pH of the reaction solution. Other examples of the additives include trace elements such as iron ions, copper ions, zinc ions, nickel ions, manganese ions, cobalt ions, and molybdenum ions.

[0017] Before supplying ammonia to the reaction solution, it is preferable to add sodium nitrate to the reaction solution to adjust the nitrate ion concentration to 2000 mg-N / L or more. Adding sodium nitrate changes the relative abundance ratio of each bacterial family, each bacterial genus, or each bacterial species measured by 16S rRNA amplicon sequencing, allowing nitrate ions to accumulate to a higher concentration.

[0018] The pH of the reaction solution is preferably adjusted to 6.0 to 8.0, preferably 6.0 to 7.0. This range improves the oxidation of nitrite by NOB, allowing a higher concentration of ammonium nitrate to accumulate in the reaction solution. Furthermore, a pH of 6.0 to 7.0 can reduce the content of undissociated ammonia and undissociated nitrite, which can adversely affect the survival and growth of AOB and NOB.

[0019] In this method for producing ammonium nitrate, an excess amount of ammonia is supplied to the reaction solution, exceeding the metabolic activity of AOB and NOB. This allows the nitric acid produced by NOB to be neutralized with ammonia to produce ammonium nitrate. This prevents the pH of the reaction solution from dropping below 5.0 due to the accumulation of nitric acid. In other words, by controlling the amount of ammonia supplied depending on the pH of the reaction solution, the pH of the reaction solution can be maintained in the range of 6.0 to 8.0, preferably 6.0 to 7.0.

[0020] On the other hand, because ammonia is oxidized to nitrite by AOB, it may be difficult to control the pH using ammonia alone (for example, when AOB activity is very high or when the amount of ammonia supplied from biomass falls below the target). In such cases, it is preferable to supply an inorganic alkali to the reaction solution to control the pH of the reaction solution. Since inorganic alkali is not metabolized by AOB, the pH of the reaction solution can be directly controlled, and the pH of the reaction solution can be easily maintained in the range of 6.0 to 8.0, preferably 6.0 to 7.0.

[0021] In the method for producing ammonium nitrate of this embodiment, the target ammonium nitrate can be accumulated in the reaction solution as long as the metabolic activity of AOB and NOB continues and as long as the supply of ammonia continues. For example, ammonium nitrate can be accumulated in the reaction solution at a concentration of 5000 mg / L or more.

[0022] Furthermore, when an inorganic alkali is added to the reaction solution, nitrate salts formed by the neutralization of nitric acid and inorganic alkali can accumulate in the reaction solution independently of the accumulation of ammonium nitrate. For example, a nitrate nitrogen (nitrate nitrogen) concentration of 5,000 mg-N / L or more can be accumulated in the reaction solution, including all nitrates other than ammonium nitrate, free nitrate ions, and ammonium nitrate. Converting this nitrate nitrogen concentration to a nitric acid concentration, 22,500 mg / L or more of nitric acid can be accumulated in the reaction solution. The reaction solution containing ammonium nitrate accumulated to a desired high concentration can be supplied for various uses, including agricultural fertilizers, including microalgae cultivation, and in the chemical industry. In this case, the high concentration of ammonium nitrate in the reaction solution provides excellent transport efficiency.

[0023] The ammonia supplied to the reaction solution may be in the form of gas or liquid. In the case of liquid, it may be aqueous ammonia dissolved in water or pure liquid ammonia. The source of the ammonia may be industrially produced ammonia or ammonia generated from biomass such as compost. One example is a method in which ammonia gas generated from compost placed in a sealed container is extracted through a gas valve and supplied to the reaction solution.

[0024] <<Apparatus for Producing Ammonium Nitrate>> A second aspect of the present invention is an apparatus for producing ammonium nitrate, comprising an ammonia supply unit, a reaction tank for holding a reaction solution containing water, AOB, and NOB, and a pH controller, wherein the ammonia supply unit supplies ammonia to the reaction tank, and the pH controller controls the amount of ammonia supplied from the ammonia supply unit in response to changes in the pH of the reaction solution, so that ammonium nitrate accumulates in the reaction solution. The production apparatus of this aspect can be used to carry out the production method of the first aspect.

[0025] An example of an embodiment of this aspect will be described with reference to Figure 1. The ammonium nitrate production apparatus 10 in Figure 1 includes an ammonia supply unit 1, a reaction tank 3 for holding the reaction liquid 2, and a pH controller 4.

[0026] An example of the ammonia supply unit 1 is compost containing manure. The components of the reaction solution 2 are the same as those explained in the first embodiment, so a duplicate explanation will be omitted here. The reaction tank 3 is a general water tank capable of holding the reaction solution 2. The electrodes of the pH controller 4 are inserted into the reaction solution 2, and constantly measure the pH of the reaction solution 2. The pH controller 4 has a function of controlling the valve of the supply pipe from the ammonia supply unit 1 to the reaction tank 3, and can automatically adjust the amount of ammonia supplied.

[0027] When ammonia is supplied from the ammonia supply unit 1, ammonium ions are generated in the reaction solution, and nitric acid (shown as nitrate ions in the diagram) is generated through the metabolic action of AOB and NOB. As the product nitric acid accumulates in the reaction solution, the pH of the reaction solution decreases. Therefore, the ammonium ions before being metabolized by AOB and the generated nitric acid are neutralized to form ammonium nitrate, which accumulates in the reaction solution. If the amount of ammonium ions supplied is relatively small compared to the metabolic action of AOB, the pH of reaction solution 2 decreases. When the pH controller 4 detects a decrease in the pH of reaction solution 2, the amount of ammonia supplied from the ammonia supply unit 1 to reaction solution 2 is increased. As a result, the pH of reaction solution 2 can be maintained within a desired range, for example, pH 6.0 to 8.0, preferably 6.0 to 7.0.

[0028] The pH controller 4 may control an inorganic alkali supply unit (not shown) in addition to the ammonia supply unit 1. An example of an inorganic alkali supply unit is a tank storing an aqueous sodium hydroxide solution. The pH controller 4 has a function of controlling a valve in the supply line from the inorganic alkali supply unit to the reaction tank 3, and can automatically adjust the supply amount of the aqueous sodium hydroxide solution supplied from the inorganic alkali supply unit in accordance with changes in the pH of the reaction solution 2. For example, if the pH of the reaction solution 2 is heading toward 5.0 or below even when the ammonia supply amount exceeds a predetermined threshold (e.g., when AOB activity is very high or when the ammonia supply amount generated in the compost drops below a certain level), the pH can be raised to neutral by supplying an aqueous sodium hydroxide solution. As a result, the pH of the reaction solution 2 can be maintained within a desired range, for example, a pH of 6.0 to 8.0, preferably 6.0 to 7.0.

[0029] <<Method for Producing Nitric Acid>> In a third aspect of the present invention, a reaction solution containing water, ammonia oxidizing bacteria (AOB), and nitrite oxidizing bacteria (NOB) is added with ammonia (NH ３ ), and the ammonia is converted to nitric acid (HNO ) by the metabolic action of AOB and NOB. ３ ), and then nitric acid is extracted from the reaction solution by electrodialysis.

[0030] The reaction solution of this embodiment and its constituent components, water, AOB, NOB and ammonia, are explained in the same manner as in the first embodiment, and therefore will not be explained again here.

[0031] In the method for producing nitric acid according to this embodiment, it is preferable to selectively remove nitric acid generated in the reaction solution as needed by electrodialysis. By removing the nitric acid as needed, accumulation of nitric acid in the reaction solution can be prevented, and the pH of the reaction solution can be prevented from dropping to, for example, 6.0 or less.

[0032] Electrodialysis is a method in which cation-exchange membranes and anion-exchange membranes are alternately placed between a positive electrode and a negative electrode to form partitions. The ions in the solution flowing between these membranes are transferred, allowing separation into a desalted stream, a stream enriched with anions, including nitrate ions, along with protons, and a stream enriched with cations, including sodium ions and ammonium ions, along with hydroxy ions. The nitric acid-enriched stream is recovered, and the ammonium ion-containing stream is returned to the reaction solution. This separation can be performed using a commercially available electrodialysis device equipped with bipolar membranes.

[0033] If the amount of nitric acid produced by the metabolic action of AOB and NOB exceeds the efficiency of nitric acid removal by electrodialysis, the pH of the reaction solution may decrease as nitric acid accumulates. As in the first embodiment, the accumulated nitric acid may be neutralized with ammonia, or an inorganic alkali may be supplied to the reaction solution to control the pH of the reaction solution. The inorganic alkali supplied to the reaction solution is removed from the reaction solution by electrodialysis, but can be recovered as a concentrated solution of inorganic alkali separated from nitric acid. The recovered concentrated inorganic alkali solution can be returned to the reaction solution as needed and used to adjust the pH of the reaction solution.

[0034] In this method for producing nitric acid, the target nitric acid can be extracted from the reaction solution by electrodialysis as long as the metabolic activity of AOB and NOB continues and as long as ammonia is supplied. The nitric acid extracted from the reaction solution can be concentrated as necessary and then supplied to various uses in agricultural fertilizers, including microalgae cultivation, and the chemical industry. By concentrating the nitric acid in advance, transportation efficiency can be improved.

[0035] <Nitric Acid Manufacturing Apparatus> A fourth aspect of the present invention is an apparatus for manufacturing nitric acid, comprising an ammonia supply unit, a reaction tank for holding a reaction solution containing water, AOB, and NOB, and an electrodialysis device, wherein the ammonia supply unit supplies ammonia to the reaction tank, and the electrodialysis device extracts nitric acid from the reaction solution.

[0036] An example of this embodiment will be described with reference to Fig. 2. The nitric acid production apparatus 20 in Fig. 2 includes an ammonia supply unit 1, a reaction tank 3 for holding the reaction liquid 2, and an electrodialysis device 5.

[0037] The explanations of the ammonia supply unit 1, the reaction solution 2, and the reaction tank 3 are the same as those of the second embodiment, and therefore will not be repeated here. The electrodialysis device 5 is not particularly limited as long as it can extract nitric acid from the reaction solution 2, and for example, a commercially available electrodialysis device equipped with a bipolar membrane can be applied. If there are ammonium ions that have been extracted from the reaction solution together with the nitric acid, they can be separated from the nitric acid and then returned to the reaction solution 2 through a return pipeline.

[0038] The nitric acid production apparatus 20 further includes an inorganic alkali supply unit 6. An example of the inorganic alkali supply unit 6 is a tank storing an aqueous sodium hydroxide solution. When necessary to adjust the pH of the reaction solution 2, an inorganic alkali is supplied from the inorganic alkali supply unit 6 through a supply pipe to the reaction tank 3. If there is any inorganic alkali that has been temporarily removed from the reaction solution together with the nitric acid, it can be separated from the nitric acid and then returned to the reaction solution 2 through a return pipe.

[0039] The nitric acid production apparatus 20 may be equipped with a pH controller (not shown). As in the above-described ammonium nitrate production apparatus 10, the pH of the reaction solution 2 is measured by the pH controller, and one or more of the valves in the supply line from the ammonia supply unit 1 to the reaction tank 3, the valves in the supply line from the inorganic alkali supply unit 6 to the reaction tank 3, and the valves in the return line of the electrodialysis device 5 are controlled in accordance with changes in pH. This allows the pH of the reaction solution 2 to be controlled to, for example, 6.0 to 8.0, preferably 6.0 to 7.0.

[0040] It is preferable to provide a filter at the suction port of the reaction solution 2 of the electrodialysis device 5 to prevent the suction of AOB and NOB dispersed in the reaction solution. It is also preferable to retain the AOB and NOB contained in the reaction solution 2 on a microorganism carrier having a diameter of, for example, about 5 to 20 mm, since this allows the mesh size of the filter installed at the suction port to be large.

[0041] The embodiments of the present invention have been described in detail above with reference to the drawings, but the specific configuration is not limited to that described above, and various design changes can be made within the scope of the gist of the present invention.

[0042] (Example 1) Experimental Objective: The objective was to demonstrate that AOB and NOB can maintain their activity in a highly nitrifying environment.

[0043] Device configuration: A lab-scale nitrification system was constructed in which the supply of ammonia water diluted to 0.5-1.0 mol / L was controlled by a pH controller. The effective volume of the nitrification reactor was 500 mL, the lower limit of pH was set to 7.0, and the temperature was set to 25°C. When the pH fell below the lower limit, the pH controller activated a peristaltic pump, and ammonia water was supplied to the nitrification reactor until the pH reached the lower limit. The nitrification reactor maintained an aerobic environment by aeration.

[0044] Materials used: A nitrifying bacterial consortium containing AOB and NOB collected from a nitrification tank at a sewage treatment plant in Omura City, Nagasaki Prefecture was used.

[0045] Experimental method: Sludge from a nitrification tank in which nitrite ions had accumulated to 300 mg-N / L through continuous supply of ammonia water was used as the inoculum. Aeration was continued with the supply of ammonia water stopped, and after confirming the growth of NOB by the decomposition of nitrite ions (Figure 3A), the supply of ammonia water was resumed (Figure 3B). When the nitrite ion concentration temporarily rose above 300 mg-N / L after the ammonia water supply was resumed, the pH was controlled at 7.0-7.5 with sodium hydroxide solution instead of ammonia water.

[0046] Results and Discussion: Nitrite ion concentration decreased rapidly between days 17 and 23 of operation, confirming the activation of NOB (Figure 3A). Subsequently, ammonia water was supplied and nitrogen concentration was monitored. The nitrate ion concentration in the reactor continued to increase, reaching approximately 6,500 mg-N / L (approximately 29,000 mg / L) (Figure 3B). The rapid increase in nitrate ion concentration suggests that both AOB and NOB were highly active and the nitrification reaction continued to proceed. It was demonstrated that high concentrations of nitrate, approaching 30,000 mg / L, could be accumulated by using sodium hydroxide, an inorganic alkali, to adjust the pH. During this nitrification reaction, sufficient ammonia water was supplied and the pH was stably controlled, so it is believed that at least a portion of the nitrate ions were neutralized by ammonia and accumulated as ammonium nitrate. Furthermore, since no nitrification inhibition was observed even when the ammonium ion concentration in the reactor reached 1000 mg-N / L, it was confirmed that ammonium nitrate of at least approximately 5700 mg / L (nitrate ion concentration 1000 mg-N / L) can be accumulated even without the use of sodium hydroxide. These results demonstrate that nitrification is possible when a nitrifying bacterial consortium is acclimatized to a high-nitrification environment by maintaining the pH within the appropriate range.

[0047] (Example 2) Experimental objective: The objective was to clarify the dynamics of free ammonia nitrogen (FAN) and free nitrite nitrogen (FNN) in a high-concentration nitrification environment.

[0048] Device configuration: A lab-scale nitrification system was constructed, controlling the supply of ammonia water diluted to 0.5-1.0 mol / L with a pH controller. A 20-L polyethylene tank (effective volume 15 L) was used as the nitrification reactor, with a pH lower limit of 7.0 and a temperature of 25°C. Two nitrification reactors were operated in parallel to accumulate high concentrations of nitrate ions. When the pH fell below the lower limit, a peristaltic pump was activated by a pH controller (SATOTECH, product name "PPH-2108"), and ammonia water was supplied to the nitrification reactor until the pH reached the lower limit. When the nitrification balance was disrupted due to an increase in nitrite ion concentration, sodium hydroxide solution was supplied to the nitrification reactor, and the supply of ammonia water was resumed when the nitrite ion concentration decreased. The nitrification reactor maintained an aerobic environment by periodically supplying air with an air pump. Nutrient solutions (calcium, chelated iron, trace elements) were supplied to the nitrification reactor at a maximum of once a week at a rate of 1 mL / L of each stock solution.

[0049] Materials used: A nitrifying bacterial consortium containing AOB and NOB collected from a nitrification tank at a sewage treatment plant in Omura City, Nagasaki Prefecture was used.

[0050] Experimental method: Nitrification reactor #1 used activated sludge immediately collected from a nitrification tank at a sewage treatment plant in Omura City, Nagasaki Prefecture. The following pretreatment was performed on the sludge to be used in nitrification reactor #2. Activated sludge immediately collected from a nitrification tank at a sewage treatment plant in Omura City, Nagasaki Prefecture, was added to a high-concentration nitrification reactor (effective volume 15 L). The nitrification reactor was operated for a certain period of time, allowing the TNN (total nitrite nitrogen) concentration to accumulate to 5023 mg-N / L and the nitrate ion concentration to accumulate to 3111 mg-N / L. This pretreated sludge with suppressed nitrite oxidation activity was used as the inoculum for nitrification reactor #2. When added to nitrification reactor #2, the sludge was diluted to a TNN concentration of 300 mg-N / L and used as the inoculum. Air was continuously supplied with the ammonia water supply stopped, and after confirming the growth of NOB by a decrease in TNN concentration, the ammonia water supply was resumed. After the supply of aqueous ammonia was resumed, when the TNN concentration temporarily increased to exceed 300 mg-N / L, the pH was controlled at 7.0-7.5 with sodium hydroxide solution instead of aqueous ammonia.

[0051] Analysis method: TAN (total ammonia nitrogen) and TNN concentrations were measured using a pack test tube, and nitrate ion concentration was measured using a nitrate ion meter (manufactured by HORIBA, trade name "LAQUAtwin"). Free ammonia nitrogen (FAN) and free nitrite nitrogen (FNN) concentrations were calculated based on the following formulas (1) and (2) using TAN or TNN and pH, assuming a temperature of 25°C.

[0052]

[0053]

[0054] In the above formulas (1) and (2), pKa is the dissociation constant at 25° C., and the pKa in formula (1) is 9.25, and the pKa in formula (2) is 3.25.

[0055] The nitrate ion production rate R in the ammonia supply engine was calculated based on the following formula (3).

[0056]

[0057] In the above formula (3), Δ[NO3- ] is the change in nitrate ion concentration during the ammonia supply period (mg-N / L), and Δt is the supply period (d).

[0058] Results and Discussion: During the ammonia supply period, the nitrate ion concentration in nitrification reactors #1 and #2 increased and peaked between days 60 and 90 of operation. In nitrification reactor #1, the concentration reached approximately 6,000-7,000 mg-N / L (Figure 4B). If the nitrate ions produced were completely neutralized with sodium hydroxide, the osmotic pressure would be approximately 0.9-1.0 Osmol / L. In nitrification reactor #2, the concentration reached approximately 7,000-8,000 mg-N / L (Figure 5B). If the nitrate ions produced were completely neutralized with sodium hydroxide, the osmotic pressure would be approximately 0.9-1.0 Osmol / L. These results demonstrate that the WWTP-derived nitrifying bacterial consortium can maintain osmotic pressures of approximately 0.9-1.1 osmol / L even under seawater-like osmotic pressure conditions (approximately 0.9-1.1 osmol / L) by reacting the generated nitrate ions with added sodium hydroxide, thereby maintaining osmotic pressures of approximately 0.9-1.1 osmol / L and enabling continuous nitrification. In nitrification reactors #1 and #2, TAN and TNN concentrations fluctuated significantly (Figures 4C and 5C). In nitrification reactor #1, the maximum TAN concentration was 829 mg-N / L. In nitrification reactor #2, the maximum TAN concentration was 1029 mg-N / L. In nitrification reactors #1 and #2, TNN concentrations were often lower than TAN concentrations. On day 57 in nitrification reactor #1 and day 51 in nitrification reactor #2, TNN concentrations rose sharply to 1489 mg-N / L. On day 1 in nitrification reactor #2, a TNN concentration of 1817 mg-N / L was observed, which is believed to be due to the high ammonia feed rate used prior to the start of the experiment. The uptake of ammonia and nitrite ions in bacterial cells can be regulated by ion channels. However, the uptake of undissociated ammonia (NH3 or FAN) and undissociated nitrite (HNO2 or FNN) in bacterial cells is not controlled by ion channels and instead passes through the cell membrane by passive diffusion. Therefore, undissociated ammonia and undissociated nitrite may cause cell damage. Non-patent document 1 discloses that FAN concentrations above 36 mg-N / L exhibit an inhibitory effect on cell activity.Non-Patent Document 2 discloses that FNN concentrations in the range of 0.22 to 2.8 mg-N / L exhibit an inhibitory effect on cell activity. The arithmetic mean FAN concentration in nitrification reactor #1 was 4.0 mg-N / L, and the arithmetic mean FAN concentration in nitrification reactor #2 was 1.0 mg-N / L. The arithmetic mean FNN concentration was 0.02 mg-N / L in both nitrification reactors #1 and #2. In this example, the FNN concentration remained well below the inhibitory concentration for most of the operation period (Figures 4D and 5D). This is likely due to the fact that the pH was controlled between 7.0 and 7.5, minimizing the proportion of undissociated TAN and TNN even at high concentrations.

[0059] (Example 3) Experimental objective: The objective was to identify nitrifying bacteria at the family or genus level contained in the sample.

[0060] Materials used: A nitrifying bacterial consortium containing AOB and NOB was used, collected from nitrification reactors #1 and 2. The samples were collected on the days elapsed since the start of operation of the nitrification reactors, as shown on the horizontal axis of FIG. 6 .

[0061] Experimental method: 16S rRNA amplicon sequencing was performed to identify nitrifying bacteria at the genus level. First, DNA from microorganisms forming the nitrifying bacterial consortium in the activated sludge sample was extracted using the ISOIL for Beads Beating Kit (Nippon Gene Co., Ltd.) according to the manufacturer's protocol. The V3-V4 region of the 16S rRNA gene was amplified from the extracted sample. Amplification was performed according to the Illumina 16S metagenomic sequencing library protocol using a universal primer pair containing the overhang sequence shown in Table 1 below: a forward primer (SEQ ID NO: 1) and a reverse primer (SEQ ID NO: 2). In Table 1 below, N represents A, T, G, or C; W represents A or T; H represents A, T, or C; and V represents A, G, or C. The DNA fragment obtained by amplifying the V3-V4 region of the 16S rRNA gene (16S rRNA V3-V4 amplicon) was purified using Ampure XP Reagent (Beckman Coulter). Index PCR was then performed using the Nextera® XT Index Kit (Illumina). The indexed PCR products were further purified and pooled in equimolar ratios. This was used as a library, and the equimolar mixture was sequenced using MiSeq (2 x 300 cycles) (commissioned by Hokkaido System Science). The data obtained by sequencing were analyzed using the QIIME2 package ver. After denoising using the DADA2 plugin (trim-left-f = 17, trim-left-r = 21, trunc-len-f = 280, trunc-len-r = 240) in 2024.10, Naive Bayes classification was performed on the SILVA database (silva-138).

[0062]

[0063] Results and Discussion: Figure 6 shows the time course of bacterial family-level relative abundance. Relative abundance is the ratio of the number of reads classified into a specific bacterial taxon (e.g., family, genus, or species) to the total number of 16S rRNA amplicon reads in a sample. The upper cluster in the heat map represents the group whose abundance increased with increasing nitrate ion concentration, while the lower cluster represents the group whose abundance decreased with decreasing nitrate ion concentration. In nitrification reactors #1 and #2, the groups whose abundance increased later during operation included the families Nitrosomonadaceae, Xanthobacteraceae, Xanthomonadaceae, and Microbacteriaceae. The Nitrosomonadaceae family includes the genus Nitrosomonas (AOB) and the genus Nitrosospira (NOB), but at the genus level, almost all were Nitrosomonas (Figures 8A and 9A). This change in proportion suggests adaptation to a high-nitrate environment. In nitrification reactor #1, Nitrospiraceae (all Nitrospira) was detected in the early stages of operation (Figures 6 and 8B). However, Nitrospira was not detected in nitrification reactor #1 after day 32 of operation, and in nitrification reactor #2 throughout the entire period. This suggests that exposure of the nitrifying bacterial consortium to high concentrations of nitrate prior to operation may have completely suppressed Nitrospira.

[0064] (Example 4) Experimental objective: The objective was to analyze closely related species of the Xanthobacteraceae family contained in the nitrifying bacteria consortium.

[0065] Results and Discussion: Figure 10 shows the relative abundance of Feature IDs within the Xanthobacteraceae family detected by 16S rRNA amplicon sequencing performed in Example 3 above. Feature IDs represent bacterial species within the Xanthobacteraceae family. To investigate whether the Xanthobacteraceae amplicon sequence variants (ASVs) detected by the 16S rRNA amplicon sequencing described above function as NOBs, a molecular phylogenetic tree was constructed using the 16S sequences of Xanthobacteraceae ASVs and known NOBs, such as the genus Nitrobacter (Figure 11). The Xanthobacteraceae ASVs obtained in nitrification reactor #1 were most closely related to Nitrobacter winogradskyi. The Xanthobacteraceae ASVs obtained in nitrification reactor #2 were most closely related to Nitrobacter hamburgensis.

[0066] (Example 5) Experimental objective: The objective was to identify nitrifying bacteria contained in the sample at the species level.

[0067] Materials used: A nitrifying bacterial consortium containing AOB and NOB was used, harvested from nitrification reactor #2 on the 84th day of operation.

[0068] Experimental Method: To identify nitrifying bacteria at the species level, full-length 16S rRNA sequencing was performed. Sequencing was performed using the PacBio Revio system according to the PacBio manual, using the universal primer pair 27F (SEQ ID NO: 3) and 1492R (SEQ ID NO: 4) shown in Table 2 below. In Table 2 below, R represents A or G, Y represents C or T, and M represents A or C. The sequencing data was processed using SMRT Link, lima, and the DADA2 denoise-ccs plugin, followed by phylogenetic analysis using the MIrROR database. Relative abundance was analyzed based on the obtained data.

[0069]

[0070] Results and Discussion: Figure 12 is a graph showing the relative abundance of bacterial species based on full-length 16S rRNA sequencing. It was confirmed that AOB was dominated by Nitrosomonas eutropha, and NOB was dominated by Nitrobacter hamburgensis.

[0071] (Example 6) Experimental objective: The objective was to evaluate acclimation methods for adapting a nitrifying bacterial consortium derived from a sewage treatment plant to a high nitrate environment.

[0072] Materials used: A nitrifying bacterial consortium containing AOB and NOB collected from a nitrification tank at a sewage treatment plant in Omura City, Nagasaki Prefecture was used.

[0073] Experimental method: A 10 L nitrification reactor was charged with 5 L of activated sludge collected from a nitrification tank at a sewage treatment plant in Omura City, Nagasaki Prefecture, Japan, 1 L of nitric acid solution (N solution), and 1 L of distilled water to create a 7 L acclimation system. 42.5 g of sodium nitrate was added to this reactor to adjust the nitrate ion concentration to 1000 mg-N / L (Nitrification Reactor #3). Similarly, 85 g of sodium nitrate was added to a 7 L acclimation system containing 7 L of effective solution to adjust the nitrate ion concentration to 2000 mg-N / L (Nitrification Reactor #4). Similarly, 127.5 g of sodium nitrate was added to a 7 L acclimation system containing 7 L of effective solution to adjust the nitrate ion concentration to 3000 mg-N / L (Nitrification Reactor #5). Nitrification reactors #3-5 were maintained in an aerobic environment by periodically supplying air via an air stone. pH was controlled as described above. Nutrient solutions (calcium, chelated iron, and trace elements) were supplied to the nitrification reactors at a rate of 1 mL / L of each stock solution at most once a week. Ammonia was supplied to the nitrification reactors. An aqueous ammonium chloride solution was used for the ammonia supply. During ammonia supply, the ammonium chloride solution was manually added at a rate that would result in an ammonia concentration of 10 mg-N / L per day relative to the effective volume of the nitrification reactor, while monitoring the nitrite ion accumulation in the nitrification reactor. Specifically, a 10 g-N / L ammonium chloride solution was prepared, and 7 mL of this solution was added per day to a reactor with an effective volume of 7 L to achieve the desired supply rate. Starting on the 49th day of operation, the ammonia concentration was increased to 50 mg-N / L per day. Starting on the 59th day, midway through the experiment, the ammonia concentration was increased from 50 mg-N / L to 100 mg-N / L per day, and the supply was continued while monitoring for the presence or absence of nitrite ion accumulation. Ammonium ion concentration was measured using the indophenol blue absorptiometry method (JIS K0102:2013). Nitrite ion concentration was measured using the naphthylethylenediamine absorptiometry method (JIS K0102 43.1.1). Nitrate ion concentration was measured using an ion chromatography system.Nitrification activity tests were conducted every 2-3 weeks using sludge collected from nitrification reactors #3-5 in an environment containing 4000 mg-N / L of sodium chloride. The nitrification activity tests were conducted as follows. First, 300 mL of collected sludge was dispensed into centrifuge tubes in approximately 25-30 mL portions. The centrifuge tubes containing the sludge were centrifuged, and the supernatant separated by the centrifugation was removed. An equal volume of saline solution to the removed supernatant was added to the centrifuge tube, and the tubes were centrifuged three times to remove unreacted ammonium ions and nitrite ions. Then, saline solution was added to the sludge separated by the centrifugal washing, and 300 mL of sludge suspension was prepared. Next, 400 mL of a solution containing 5000 mg-N / L sodium nitrate and 125 mg-N / L ammonium ions and 100 mL of sludge were added to a plastic bottle-type nitrification reactor (effective volume 500 mL) (AOB activity test area) so that the nitrate nitrogen concentration in the reactor after mixing was 4000 mg-N / L and the ammonium ion concentration was 100 mg-N / L. Ammonium chloride was used as the ammonium ion. 400 mL of a solution containing 5000 mg-N / L sodium nitrite and 125 mg-N / L nitrite ions and 100 mL of sludge were added to a plastic bottle-type nitrification reactor (effective volume 500 mL) (NOB activity test area) so that the nitrate nitrogen concentration in the reactor after mixing was 4000 mg-N / L and the nitrite ion concentration was 100 mg-N / L. Sodium nitrite was used as the nitrite ion. The two flasks were subjected to the same pH control and aerobic conditions as those in nitrification reactors #3-5. The time course data for ammonium and nitrite ion concentrations were fitted to a first-order reaction model, and the reaction rate constant k was calculated using the least squares method.

[0074] Results and Discussion: Figure 13 shows the nitrogen dynamics in the nitrification reactors under different initial nitrate nitrogen conditions. In nitrification reactor #3, the nitrite ion concentration accumulated to a maximum of 14.9 mg-N / L (the FNN concentration calculated using the above formula (2) was 0.025 mg-N / L) on the fifth day of operation. When ammonia supply to nitrification reactor #3 was subsequently stopped for two days, the nitrite ion concentration decreased. Even after this, the nitrite ion concentration remained below 5 mg-N / L, even when ammonia supply to nitrification reactor #3 was continued. Meanwhile, in nitrification reactors #4 and #5, which had higher initial nitrate ion concentrations, the nitrite ion accumulation observed at the initial stage continued compared to nitrification reactor #3. In nitrification reactor #4, the nitrite ion concentration accumulated to a maximum of 100.2 mg-N / L (the FNN concentration calculated using the above formula (2) was 0.17 mg-N / L). In nitrification reactor #5, the nitrite ion concentration accumulated up to a maximum of 139.9 mg-N / L (the FNN concentration calculated by the above formula (2) was 0.24 mg-N / L). After the 20th day of operation, no nitrite ion accumulation was observed in nitrification reactors #3-5. This suggests that NOB began to adapt to the nitrification reactor environment after the 20th day of operation. Even after the 49th day of operation, when the ammonia feed rate increased, no nitrite ion accumulation was observed, and the nitrification reaction continued smoothly. After the 70th day of operation, the nitrite ion concentration in nitrification reactor #3 was 3310 mg-N / L, the nitrite ion concentration in nitrification reactor #4 was 4954 mg-N / L, and the nitrite ion concentration in nitrification reactor #5 was 6784 mg-N / L. Figure 14 shows the time course of the reaction rate constant, which is an indicator of the nitrification activity (ammonia and nitrite oxidation ability) of the nitrifying bacteria in nitrification reactors #3-5. The reaction rate constant k estimated using a first-order reaction model increased from the middle to the latter stages of operation. An increase in the reaction rate constant k indicates a more advanced oxidation reaction, suggesting high oxidation activity. These results revealed that the nitrifying bacteria contained in the sludge from the sewage treatment plant adapt to a high-nitrate environment over approximately 35-70 days. Among nitrification reactors #3-5, nitrification reactor #5 had the highest ammonia oxidation activity, followed by nitrification reactor #4, and nitrification reactor #3 had the lowest.Among nitrification reactors #3-5, nitrification reactor #4 had the highest nitrite oxidation activity, followed by nitrification reactor #3, and nitrification reactor #5 had the lowest. These results revealed that ammonia oxidation activity and nitrite oxidation activity show different trends. Furthermore, it was revealed that acclimating sludge to an initial nitrate ion concentration of 2000 mg-N / L or higher is effective in accumulating high concentrations of nitrate ions. Furthermore, it was suggested that AOB is more adaptable to high-nitrate environments than NOB.

[0075] (Example 7) Experimental Objective: The objective was to analyze the temporal changes in the relative abundance of nitrifying bacteria at the family or genus level in a high nitrate environment.

[0076] Materials used: Nitrifying bacterial consortia containing AOB and NOB collected from nitrification reactors #3-5 were used. The samples were collected on the days elapsed since the start of operation of the nitrification reactors, as shown on the horizontal axis of Figures 15-17.

[0077] Experimental method: 16S rRNA amplicon sequencing was performed as described above.

[0078] Results and Discussion: Figures 15-17 show the time course of the relative abundance of each genera within the Nitrosomonadaceae, Nitrospiraceae, and Xanthobacteraceae families in nitrification reactors #3-5. In nitrification reactor #3, the relative abundance of Nitrospiraceae (all Nitrospira genus) on day 0 (start of operation) was 5.7%, and the relative abundance of bacteria closely related to NOB within the Xanthobacteraceae family was 0%. In nitrification reactor #4, the relative abundance of Nitrospiraceae (all Nitrospira genus) on day 0 (start of operation) was 5.7%, and the relative abundance of bacteria closely related to NOB within the Xanthobacteraceae family was 0%. In nitrification reactor #5, the relative abundance of Nitrospiraceae (all Nitrospira genus) on day 0 (start of operation) was 5.7%, and the relative abundance of bacteria closely related to NOB within the Xanthobacteraceae family was 0%. In nitrification reactors #4 and #5, which were operated in a high-nitrate environment, the relative abundance of Nitrospira (NOB) decreased and the relative abundance of Xanthobacteraceae bacteria, which are closely related to Nitrosomonas (AOB) and Nitrobacter (NOB), increased as the nitrate ion concentration increased over the course of operation. These results suggest that acclimation to an initial nitrate ion concentration of 2000 mg-N / L or higher promotes the dominance of Nitrosomonas and Xanthobacteraceae bacteria, which are adaptable to high-nitrate environments.

[0079] Nitrospira, also known as NOB, was the only nitrifying bacterium detected in nitrification reactor #1, which had the lowest nitrate ion concentration, up to day 37 of operation. This suggests that Nitrospira may be a complete ammonia-oxidizing bacterium (COMAMMOX), capable of oxidizing both ammonia and nitrite.

[0080] (Example 8) Experimental objective: The objective was to analyze closely related species of the Xanthobacteraceae family contained in the nitrifying bacteria consortium.

[0081] Results and Discussion: Figure 18 shows the relative abundance of Feature IDs within the Xanthobacteraceae family detected by 16S rRNA amplicon sequencing in Example 7. Feature IDs represent bacterial species within the Xanthobacteraceae family. To investigate whether the Xanthobacteraceae amplicon sequence variants (ASVs) detected by the 16S rRNA amplicon sequencing described above function as NOBs, a molecular phylogenetic tree was created using the 16S sequences of Xanthobacteraceae ASVs and known NOB species, the Nitrobacter genus (Figure 19). The Xanthobacteraceae ASVs obtained in nitrification reactor #3 were suggested to be closely related to species such as Nitrobacter vulgaris. The Xanthobacteraceae ASVs obtained in nitrification reactors #4 and #5 were most closely related to Nitrobacter winogradskii.

[0082] 1... Ammonia supply unit, 2... Reaction liquid, 3... Reaction tank, 4... pH controller, 5... Electrodialysis device, 6... Inorganic alkali supply unit, 10... Ammonium nitrate manufacturing device, 20... Nitric acid manufacturing device

Claims

1. A method for producing ammonium nitrate, comprising: supplying ammonia to a reaction solution containing water, ammonia-oxidizing bacteria, and nitrite-oxidizing bacteria including at least bacteria belonging to the family Xanthobacteraceae; converting the ammonia into nitrate through metabolic action of the ammonia-oxidizing bacteria and the nitrite-oxidizing bacteria; generating ammonium nitrate through a neutralization reaction between the ammonia and the nitrate; and accumulating the ammonium nitrate in the reaction solution.

2. The method for producing ammonium nitrate according to claim 1, wherein sodium nitrate is added to the reaction solution before supplying ammonia to the reaction solution, so that the nitrate ion concentration in the reaction solution is 2000 mg-N / L or more.

3. The method for producing ammonium nitrate according to claim 1, wherein the proportion of reads classified as Nitrospiraceae in the reaction solution at the start of ammonium nitrate production, as measured by 16S rRNA amplicon sequencing, is 6% or less of the total number of 16S rRNA amplicon reads in the reaction solution.

4. The method for producing ammonium nitrate according to claim 1, wherein the pH of the reaction solution is maintained in the range of 6.0 to 8.

0.

5. The method for producing ammonium nitrate according to claim 4, wherein an excess amount of ammonia exceeding the metabolic activity is supplied to the reaction solution.

6. The method for producing ammonium nitrate according to claim 5, wherein an inorganic alkali is supplied to the reaction solution.

7. The method for producing ammonium nitrate according to claim 1, wherein the supply of ammonia is continued until the concentration of nitrate nitrogen accumulated in the reaction solution reaches 5000 mg-N / L or more.

8. The method for producing ammonium nitrate according to claim 1, wherein the supply of ammonia is continued until the concentration of ammonium nitrate accumulated in the reaction solution reaches 5000 mg / L or more.

9. A method for producing nitric acid, comprising: supplying ammonia to a reaction solution containing water, ammonia-oxidizing bacteria, and nitrite-oxidizing bacteria; converting the ammonia into nitric acid through the metabolic action of the ammonia-oxidizing bacteria and the nitrite-oxidizing bacteria; and extracting the nitric acid from the reaction solution by electrodialysis.

10. The method for producing nitric acid according to claim 9, wherein an inorganic alkali is added to the reaction solution to adjust the pH of the reaction solution to 6.0 to 8.

0.

11. The method for producing nitric acid according to claim 10, wherein the inorganic alkali is extracted together with nitric acid from the reaction solution by the electrodialysis method, and the extracted inorganic alkali is returned to the reaction solution.

12. An apparatus for producing ammonium nitrate, comprising: an ammonia supply unit; a reaction tank for holding a reaction solution containing water, ammonia oxidizing bacteria, and nitrite oxidizing bacteria; and a pH controller, wherein the ammonia supply unit supplies ammonia to the reaction tank; and the pH controller controls the amount of ammonia supplied from the ammonia supply unit in accordance with changes in the pH of the reaction solution, so that ammonium nitrate accumulates in the reaction solution.

13. The apparatus for producing ammonium nitrate according to claim 12, further comprising an inorganic alkali supply unit that supplies inorganic alkali to the reaction tank, wherein the pH controller also controls the amount of inorganic alkali supplied from the inorganic alkali supply unit in accordance with changes in pH of the reaction solution.

14. The apparatus for producing ammonium nitrate according to claim 12 or 13, wherein the pH of the reaction solution is controlled to be 6.0 to 8.0 by the control of the pH controller.

15. A nitric acid manufacturing apparatus comprising an ammonia supply unit, a reaction tank for holding a reaction solution containing water, ammonia oxidizing bacteria and nitrite oxidizing bacteria, and an electrodialysis device, wherein the ammonia supply unit supplies ammonia to the reaction tank, and the electrodialysis device extracts nitric acid from the reaction solution.

16. The nitric acid manufacturing apparatus according to claim 15, wherein the electrodialysis device extracts ammonium ions together with nitric acid from the reaction solution and returns the extracted ammonium ions to the reaction tank.

17. The nitric acid manufacturing apparatus according to claim 15 or 16, further comprising an inorganic alkali supply unit that supplies an inorganic alkali to the reaction tank, wherein the electrodialysis device extracts the inorganic alkali together with nitric acid from the reaction solution and returns the extracted inorganic alkali to the reaction tank.

18. The nitric acid manufacturing apparatus according to claim 17, further comprising a pH controller, which controls the amount of ammonia supplied from the ammonia supply unit, the amount of inorganic alkali supplied from the inorganic alkali supply unit, or the amount of ammonium ions or inorganic alkali returned from the electrodialysis device in accordance with changes in the pH of the reaction solution, so that the pH of the reaction solution is 6.0 to 8.0.

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