Water purification agents and water purification methods

Abstract

To provide a water purification agent and a water purification method that can denitrify rearing water in an aerobic environment. [Solution] The water purification agent is a water purification agent for purifying the water used to raise ornamental fish, and contains Pseudomonas bacteria as aerobic denitrifying bacteria. The water purification method involves adding the water purification agent containing Pseudomonas bacteria as aerobic denitrifying bacteria to a tank in which water used to raise ornamental fish is stored.

Description

Technical Field

[0001] The present invention relates to a water purification agent capable of denitrifying breeding water and a water purification method. Background Art

[0002] When ornamental fish are bred, the breeding water in the aquarium gradually becomes dirty, and a phenomenon in which algae and the like adhere to the inner wall surface of the aquarium is observed. Such a phenomenon not only hinders the viewing of the ornamental fish, but also deteriorates the living environment of the ornamental fish. In addition, algae and the like that have once adhered require physical removal means such as wiping off, which is extremely hard work especially in large aquariums and the like. In order to prevent such a phenomenon, purifying the breeding water is an important issue.

[0003] Normally, the breeding water in the aquarium is circulated, and the circulated water is treated with a filter medium or the like to remove suspended solids and organic matter in the water. The filter medium is effective for the purpose of removing solid contaminants. However, nitrogen content that increases due to feed, excrement, and the like is converted into nitrous acid and nitrate ions by the action of aerobic bacteria and dissolved in water, so the nitrogen content (nitrous acid and nitrate ions) cannot be removed by the filter medium.

[0004] As a method for removing nitrogen content, a method of denitrifying by denitrifying bacteria in an anaerobic environment is common. Denitrification occurs due to the activity of anaerobic bacteria, but the oxygen concentration is high in the environment inside an aquarium where ornamental fish are bred, making it difficult to create an anaerobic environment. In order to denitrify breeding water, it is necessary to introduce large-scale equipment (for example, Patent Document 1) or replace the breeding water periodically. Prior Art Documents Patent Documents

[0005] Patent Document 1 Japanese Unexamined Patent Application Publication No. 2022-066374 Summary of Invention Problems to be Solved by the Invention

[0006] However, installing large-scale denitrification equipment incurs significant costs. Regularly changing the aquarium water is also time-consuming for the aquarist. Therefore, there is a need for a water purification agent that can denitrify aquarium water in an aerobic environment.

[0007] The purpose of this disclosure is to provide a water purification agent and a water purification method that can denitrify rearing water in an aerobic environment. [Means for solving the problem]

[0008] The water purification agent disclosed herein is a water purification agent for purifying the water used to raise ornamental fish, and contains Pseudomonas bacteria as aerobic denitrifying bacteria.

[0009] The water purification method disclosed herein involves adding a water purification agent containing Pseudomonas bacteria as aerobic denitrifying bacteria to a tank in which water for raising ornamental fish is stored. [Brief explanation of the drawing]

[0010] [Figure 1] Figure 1 is a diagram illustrating the water purification test. [Figure 2] Figure 2 is a graph showing the changes in ammonia concentration during the water purification test. [Figure 3] Figure 3 is a graph showing the changes in nitrite concentration during the water purification test. [Figure 4] Figure 4 is a graph showing the changes in nitrate concentration during the water purification test. [Modes for carrying out the invention]

[0011] <Water purification agent> Water purification agents are used to purify the water used to keep ornamental fish. Ornamental fish include freshwater and saltwater fish that can be kept in captivity, such as medaka, goldfish, and tropical fish. The water purification agent is added to the tank in which the fish are kept.

[0012] The water purification agent contains aerobic denitrifying bacteria. With this composition, adding the water purification agent to an aquarium for keeping ornamental fish allows for denitrification of the aquarium water in an aerobic environment. This eliminates the need for large-scale denitrification equipment and regular water changes, reducing the burden on the aquarist. The number of aerobic denitrifying bacteria contained in the water purification agent is 1.0 × 10⁻⁶. 6 It is preferable that the cfu / g level is 1 or higher.

[0013] Water purification agents may contain bacteria other than aerobic denitrifying bacteria. Preferably, water purification agents contain, for example, Bacillus bacteria. Preferably, the number of aerobic denitrifying bacteria in the water purification agent is less than or equal to the number of Bacillus bacteria in the water purification agent, and preferably 20% or more of the number of Bacillus bacteria in the water purification agent.

[0014] Aerobic denitrifying bacteria are microorganisms that can perform denitrification even in aerobic environments. Examples of aerobic denitrifying bacteria include Pseudomonas, Alcaligenes, and Paracoccus species. Water purification agents may contain multiple species of aerobic denitrifying bacteria.

[0015] Aerobic denitrifying bacteria are preferably of the genus Pseudomonas. This is because some species of Pseudomonas can function even in anaerobic environments, have high metabolic capacity, and possess the ability to decompose various organic substances.

[0016] Examples of Pseudomonas species include Pseudomonas mosselii, Pseudomonas stutzeri, and Pseudomonas resinovorans.

[0017] The aerobic denitrifying bacterium is preferably Pseudomonas mosseri. This configuration improves the denitrification capacity of the aquarium water by the water purification agent. Further details will be provided later.

[0018] The aerobic denitrifying bacteria are preferably in a powder form. Thereby, the aerobic denitrifying bacteria can be stably stored. The powder of the aerobic denitrifying bacteria is obtained, for example, by freeze-drying the aerobic denitrifying bacteria.

[0019] The water purification agent may be formed into a tablet form, a liquid form, a powder form, or the like. The water purification agent is preferably formed into a tablet form by mixing a carrier supporting the aerobic denitrifying bacteria and an excipient. According to such a configuration, the water purification agent can be directly put into water, and the labor of measuring powder or liquid, the labor of cleaning when it falls out of the water tank, and the discomfort when it adheres to hands can be reduced.

[0020] The tablet form is, for example, a cubic, cuboid, spherical, cylindrical, prismatic, conical, pyramidal, ellipsoidal, circular cylindrical, or a combined tablet shape thereof. From the viewpoints of moldability and usability, the water purification agent is preferably formed into a substantially triangular tablet shape.

[0021] The carrier is composed of a carrier base material and the (powder of the) aerobic denitrifying bacteria. The carrier base material is preferably an inorganic substance, and is more preferably an inorganic mineral salt from the viewpoints of moldability, storage stability and water solubility. The inorganic mineral salt is preferably sodium sulfate or magnesium sulfate, which is easily soluble in water and has little effect on liquid property (pH). In particular, the carrier base material is preferably sodium sulfate which has no effect on the total hardness of water. The carrier base material may be an organic substance.

[0022] The aerobic denitrifying bacteria are preferably contained in a weight ratio of 10% or more in the carrier, more preferably 20% or more. The carrier base material is preferably contained in a weight ratio of 80% or more in the carrier, more preferably 90% or more.

[0023] The excipient is preferably an inorganic substance, non-foaming, easily soluble in water, and has little effect on the pH of the solution. It may be sodium sulfate, or a compound of sodium sulfate and magnesium sulfate. From the viewpoint of minimizing the effect on the total hardness of the water, it is preferable that the weight ratio of magnesium sulfate in the total water purification agent be 10% or less. This can suppress the increase in the total hardness of the water. In addition, by including magnesium sulfate, the amount of sodium can be reduced, and the increase in the salinity of the water can be suppressed. The excipient may also be an organic substance.

[0024] The excipient may be any one or a combination of any of the following: sodium bicarbonate (potassium bicarbonate, potassium carbonate, sodium citrate, etc. are also acceptable), sodium carbonate (potassium carbonate, sodium sesquicarbonate, etc. are also acceptable), sodium sulfate (potassium sulfate, sodium dihydrogen phosphate, trisodium citrate, etc. are also acceptable), fumaric acid (citric acid, succinic acid, etc. are also acceptable), and sorbitol (sugar alcohols such as mannitol, xylitol, and maltitol are also acceptable). When combining multiple components, the proportions of each component in the excipient are preferably, for example, 40% to 60% of sodium bicarbonate, 10% to 20% of sodium carbonate, 5% to 15% of sodium sulfate, 5% to 15% of fumaric acid, and 0.2% to 1.0% of sorbitol. Other possible combinations include 20%-30% mannitol, 15%-25% microcrystalline cellulose, 3%-7% croscarmellose sodium, 0.5%-1.0% magnesium stearate, 1%-3% sodium ascorbate, and 5%-13% zeolite powder. Other possible combinations include 10%-70% sodium sulfate and 20%-30% mannitol.

[0025] When aerobic denitrifying bacteria are obtained as a powder by freeze-drying, the water purification agent preferably contains a protective agent. The protective agent is a combination of one or more of the following: sugars such as trehalose, sucrose, lactose, and dextran (vitrification and protection of cell membranes); polymers such as polyvinylpyrrolidone (PVP), hydroxyethyl starch (HES), and gelatin (membrane stabilization and improved drying resistance); amino acids and proteins such as glycine, proline, and gelatin hydrolysate (collagen peptide) (membrane stabilization and antioxidant); glycerol, PEG (polyethylene glycol), milk components (skim milk powder, whey protein), ascorbic acid (vitamin C), sodium lactate, glutathione, and sodium glutamate.

[0026] The protective agent may contain, for example, 5% to 10% trehalose, 1% to 3% PVP, and 0.5% to 1.0% gelatin as a general composition for freeze-drying. The protective agent may also contain, for example, 5% to 10% sucrose, 1% to 3% HES, 1% to 2% dextran (40kDa), and 0.2% to 0.5% glutathione as a composition for long-term storage.

[0027] The protective agent may contain, for example, 5% to 8% trehalose, 2% to 3% PVP, 0.1% to 0.5% ascorbic acid, and 0.2% to 0.5% glutathione to improve oxidative stress resistance (for environmentally harsh conditions). The protective agent may contain, for example, 5% to 15% glycerol, 2% to 5% trehalose, 2% to 5% PEG, and 1% to 2% HES for temperature-controlled storage. The protective agent may contain 5% to 10% sucrose and 0.1% to 0.5% gelatin from a cost-reduction perspective. It may also contain 3% to 5% skim milk and 3% to 5% trehalose. It may also contain 3% to 5% skim milk and 3% to 5% sucrose. It may contain 3% to 5% skim milk, 3% to 5% trehalose or sucrose, and 3% to 5% monosodium glutamate.

[0028] <Method for preparing water purification agents> The following describes an example of a method for producing a water purification agent. However, this does not limit the method of producing the water purification agent; water purification agents produced by methods or under conditions other than those described below are also included in this invention.

[0029] (Structure of aerobic denitrifying bacteria) (1) Collect water from a river or lake and use that water (aquarium water) to raise ornamental fish such as goldfish using common methods. The river or lake from which the water is collected should preferably have a rich biodiversity and not be polluted by wastewater. (2) Observe the change in nitrate concentration in the rearing water over time, and if a decrease in nitrate concentration is observed, culture the bacteria contained in the rearing water using a selective medium for denitrifying bacteria (Giltay medium). Culture is performed in a constant temperature bath at 30°C with oxygen present, and bacteria that can be active in an aerobic environment are selected. (3) Isolate and culture the colonies that appear on the selective medium for denitrifying bacteria to establish a strain of aerobic denitrifying bacteria.

[0030] The established bacterial strains can be identified, for example, by genetic analysis using the Sanger method. For instance, Pseudomonas mosselii can be obtained from water samples collected from rivers and lakes. The obtained Pseudomonas mosselii strain was deposited with the National Institute of Technology and Evaluation (NPMD) Patent Microorganism Depository Center (NPMD) (2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture, Japan) on March 4, 2025. The deposit number is NITE p-04298.

[0031] (Powder of aerobic denitrifying bacteria) The aerobic denitrifying bacteria strains obtained above can be collected using a centrifuge or similar device, residual culture medium can be removed, and the mixture can be freeze-dried using a known method. A protective agent can then be added to obtain aerobic denitrifying bacteria powder. In particular, Pseudomonas bacteria can be stored stably by freeze-drying. The aerobic denitrifying bacteria powder may be mixed with other powders (e.g., zeolite or calcium carbonate) to increase its volume.

[0032] (Carrier) By mixing the aerobic denitrifying bacteria powder obtained above with a support substrate in any ratio, the aerobic denitrifying bacteria are supported on the support substrate. The number of aerobic denitrifying bacteria is then 1.0 × 10⁶. 6 It is preferable that the cfu / g level is 1 or higher.

[0033] If the carrier is large (for example, 1 mm or larger), it is preferable to crush the carrier with a mixer or pulverizer to improve tableting performance, as described later. It is preferable to mix the aerobic denitrifying bacteria powder and the carrier material in a weight ratio of 3:7 to 2:8.

[0034] (Mixing of carrier and excipient) The carrier and excipient obtained above are placed in a thick plastic bag or powder mixer in a weight ratio of 0.1 to 9.9 to 2 to 8, and mixed. To form tablets of sufficient hardness, when using a carrier with magnesium sulfate as the base material, it is preferable to mix the carrier and excipient in a weight ratio of 1 (or less) to 9 (or more). When using a carrier with sodium sulfate as the base material, it is preferable to mix the carrier and excipient in a weight ratio of 2 to 8. If the powders solidify after mixing the carrier and excipient, it is preferable to remove the solidified powder using a 1 mm mesh screen from the viewpoint of improving tablet compressibility, as described later.

[0035] (Tablet compression) The mixture of the carrier and excipient obtained above can be compressed into tablets using a tablet press to obtain a tablet-shaped water purification agent. The number of aerobic denitrifying bacteria contained in the water purification agent (1 tablet) is 1.0 × 10⁶. 6 It is preferable that the cfu / g level is 10 or higher. It is preferable to measure the number of bacteria contained in each tablet of the water purification agent in batches of 10 tablets. This is because if the weight of each tablet of the water purification agent is measured individually, it is difficult to determine whether the measured value is within the margin of error.

[0036] <Water purification methods> The water purification method includes the step of introducing a water purification agent containing Pseudomonas bacteria as aerobic denitrifying bacteria into a tank in which aquarium water for raising ornamental fish is stored. By introducing the water purification agent into the aquarium in which ornamental fish are raised, denitrification of the aquarium water becomes possible in an aerobic environment. [Examples]

[0037] <Example 1> As an example, a water purification agent prepared by isolating and culturing Pseudomonas mosselii (deposit number NITE p-04298) was added to physiological saline to prepare a 20 mL suspension. The number of Pseudomonas mosselii bacteria in the suspension was 1.0 × 10⁶. 6 It is cfu / g.

[0038] <Example 2> As Example 2, a water purification agent prepared by isolating and culturing Pseudomonas stutzeri (deposit number FERM p-17713, National Institute of Advanced Industrial Science and Technology) was added to physiological saline to prepare a 20 mL suspension. The number of Pseudomonas stutzeri bacteria in the suspension was 1.0 × 10⁶. 6 It is cfu / g.

[0039] <Example 3> As Example 3, a water purification agent prepared by isolating and culturing Pseudomonas resinovorans (deposit number NBRC 113413, National Institute of Technology and Evaluation) was added to physiological saline to prepare a 20 mL suspension. The number of Pseudomonas resinovorans bacteria in the suspension was 1.0 × 10⁶. 6 It is cfu / g.

[0040] <Water quality purification test> Four tank sets were prepared, each containing 10 liters of chlorine-neutralized tap water (see Figure 1), with a hang-on filter (GEX Slim Filter SS) installed. One goldfish (weighing approximately 3.5 g) was placed in each tank. Three of the tank sets contained one of the fungi from Examples 1-3, while the remaining tank set was left blank (comparative example: blank). To prevent contamination by airborne bacteria, glass covers (see Figure 1) were placed over each tank 1, and a water purification test was conducted in a constant temperature chamber at 25°C.

[0041] Then, in each aquarium setup, goldfish were fed 0.1g / day of goldfish flake food (GEX Kingyo Genki Probiotic Flakes) and raised, and the concentrations of ammonia, nitrite, and nitrate were measured every 7 days. Figure 2 is a graph showing the changes in ammonia concentration, Figure 3 is a graph showing the changes in nitrite concentration, and Figure 4 is a graph showing the changes in nitrate concentration.

[0042] As shown in Figures 2 to 4, the concentrations of ammonia, nitrite, and nitrate in each tank set remained roughly the same until day 7. However, after day 7, the ammonia concentration in Example 1 decreased more rapidly than the others. Furthermore, in Example 1, the concentrations of nitrite and nitrate increased more rapidly than the others, and decreased more rapidly after day 21 compared to the others.

[0043] The nitric acid concentration in Examples 1-3 decreased on day 35, while there was no decrease in nitric acid concentration in the comparative example. In particular, the nitric acid concentration in Example 1 was about half the concentration of the others on day 35.

[0044] The results of the water purification tests confirmed that adding the water purification agents according to Examples 1 to 3 has the effect of lowering nitrate concentration (denitrification capacity in an aerobic environment). In particular, when the water purification agent according to Example 1 was added, the concentration peak of nitrate in the aquarium set was lowered and the decrease in nitrate concentration was faster compared to when other water purification agents were added (high denitrification capacity in an aerobic environment). Furthermore, when the water purification agent according to Example 1 was added, the concentrations of ammonia and nitrite in the aquarium set decreased faster compared to when other water purification agents were added, confirming that it also possesses nitrification capacity in an aerobic environment.

[0045] [1] The water purification agent disclosed herein is a water purification agent for purifying the water used to raise ornamental fish, and contains Pseudomonas bacteria as aerobic denitrifying bacteria. With this configuration, by adding the water purification agent to an aquarium in which ornamental fish are kept, denitrification of the water becomes possible in an aerobic environment.

[0046] [2] In the water purification agent described in [1] above, it is preferable that the aerobic denitrifying bacterium is Pseudomonas mosselii. With such a configuration, the denitrification capacity of the aquarium water by the water purification agent can be improved.

[0047] [3] In the water purification agent described in [1] or [2] above, it is preferable that the aerobic denitrifying bacteria are in powder form. With such a configuration, the aerobic denitrifying bacteria can be stored stably.

[0048] [4] The water purification agent described in [3] above preferably includes a protective agent for protecting aerobic denitrifying bacteria.

[0049] [5] The water purification agent described in any one of the above [1] to [4] is preferably formed in tablet form by mixing a carrier on which aerobic denitrifying bacteria are supported with an excipient. With such a configuration, the water purification agent can be added directly to the water, reducing the effort of measuring powders or liquids, the effort of cleaning up if it falls outside the tank, and the unpleasant feeling of it getting on your hands.

[0050] [6] The water purification method disclosed herein involves introducing a water purification agent containing Pseudomonas bacteria as aerobic denitrifying bacteria into a tank in which water for raising ornamental fish is stored. By introducing the water purification agent into a tank in which ornamental fish are raised, denitrification of the water becomes possible in an aerobic environment.

[0051] [7] In the water purification method described in [4] above, it is preferable that the aerobic denitrifying bacterium is Pseudomonas mosselii. This method makes it possible to improve the denitrification capacity of the aquarium water by the water purification agent.

[0052] [8] In the water purification method described in [6] or [7] above, it is preferable that the aerobic denitrifying bacteria are in powder form. This method allows for stable storage of the aerobic denitrifying bacteria.

[0053] [9] In the water purification method described in [8] above, it is preferable that the water purification agent includes a protective agent for protecting aerobic denitrifying bacteria.

[0054]

[10] In the water purification method described in any one of the above [6] to [9], it is preferable that the water purification agent is formed into a tablet shape by mixing a carrier on which aerobic denitrifying bacteria are supported with an excipient. With such a method, the water purification agent can be added directly to the water, reducing the effort of measuring powders or liquids, the effort of cleaning up if it falls outside the tank, and the unpleasant feeling of it getting on your hands.

[0055] While embodiments of the water purification agent and water purification method relating to this disclosure have been described, it should be understood that the specific configuration is not limited to these embodiments. The scope of this disclosure is indicated not only by the above-described embodiments but also by the claims, and furthermore, all modifications within the meaning and scope of equivalence to the claims.

[0056] The water purification agent and water purification method relating to this disclosure are not limited in any way to the embodiments described above, nor are they limited to the effects described above. The water purification agent and water purification method relating to this disclosure can be modified in various ways without departing from the spirit of the disclosure. Furthermore, it is possible to arbitrarily combine and adopt each of the components adopted in the embodiments described above. [Explanation of Symbols]

[0057] 1...Aquarium, 2...Glass cover

Claims

1. A water purification agent for purifying the water used to keep ornamental fish, A water purification agent characterized by containing Pseudomonas bacteria as aerobic denitrifying bacteria.

2. The water purification agent according to claim 1, characterized in that the Pseudomonas species is Pseudomonas mosselii.

3. The water purification agent according to claim 1, characterized in that the aerobic denitrifying bacteria are in powder form.

4. The water purification agent according to claim 3, characterized by comprising a protective agent for protecting the aerobic denitrifying bacteria.

5. A water purification agent according to any one of claims 1 to 4, characterized in that it is formed into a tablet shape by mixing a carrier on which the aerobic denitrifying bacteria are supported with an excipient.

6. A method for purifying water, characterized by adding a water purification agent containing Pseudomonas bacteria as aerobic denitrifying bacteria to a tank in which water for raising ornamental fish is stored.

7. The water purification method according to claim 6, characterized in that the Pseudomonas species is Pseudomonas mosselii.

8. The water purification method according to claim 6, characterized in that the aerobic denitrifying bacteria are in powder form.

9. The water purification method according to claim 8, characterized in that the water purification agent includes a protective agent for protecting the aerobic denitrifying bacteria.

10. The water purification method according to any one of claims 6 to 9, characterized in that the water purification agent is formed into a tablet shape by mixing a carrier on which the aerobic denitrifying bacteria are supported with an excipient.

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