How to remove biofilm

Abstract

To provide a biofilm removal method that can effectively remove a biofilm formed in a water system such as devices and facilities including a water-contacting member such as a water-cooling tower, bathtub piping.SOLUTION: This invention is a biofilm removal method in which a metal-containing biofilm formed by a water system is brought into contact with (a) a compound capable of producing hydroxyl radicals and (b) a reducing agent.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for removing a biofilm that has developed in an aqueous system. [Background technology]

[0002] Biofilms, also called biofilms or slimes, generally refer to structures formed by microorganisms such as bacteria that are produced by microorganisms and grow on the surface of a material, and that encase the microorganisms with polymeric substances such as polysaccharides, proteins, and nucleic acids. When biofilms are formed, various problems occur due to microorganisms, and they are problematic in various industrial fields. For example, when biofilms are formed in the piping of a food plant, the biofilms may peel off and become mixed into the product as foreign matter, or may cause food poisoning due to toxins derived from the bacteria. Furthermore, biofilm formation on metal surfaces may cause metal corrosion and accelerate the aging of equipment.

[0003] Conventionally, systems using aqueous media, such as cooling systems for factory equipment and buildings using water-cooled cooling towers, and cooling pools, have been used. In such systems, microbial contamination of the water used is a problem. For example, in a cooling system using a water-cooled cooling tower, microorganisms mixed in the cooling water form biofilms in the piping, especially in the heat exchanger. The biofilm formed in the heat exchanger reduces the efficiency of heat exchange and increases the power consumption of the cooling system. Periodic water replacement and cleaning are required to prevent biofilm formation, but these increase the maintenance costs of the system. In facilities and devices that use cooling water such as water-cooled cooling towers, microbial bactericides and growth inhibitors called slime control agents are sometimes added to the cooling water to suppress biofilms, but from the viewpoints of safety, corrosion of metal piping, and chemical costs, treatment without using slime control agents is preferable.

[0004] Various proposals have been made so far as measures against contamination caused by microorganisms. US Patent No. 5,399,633 discloses that disinfecting and decontaminating agents including vitamins, metal ions, surface active compounds, and certain antimicrobial agents can be used to break down biofilms. Patent Document 2 discloses a water treatment method for preventing biofilms in a cooling water system in which cooling water cooled in a cooling tower is circulated through a heat exchanger and make-up water is supplied to the cooling tower, the water treatment method removing phosphoric acid from the make-up water and using a non-phosphorus-based corrosion inhibitor as a corrosion inhibitor. Patent Document 3 discloses a biofilm remover composition for hard surfaces, which contains 1 mass % or more and 40 mass % or less of an internal olefin sulfonate. Patent Document 4 discloses a composition for inhibiting biofilm formation, which contains a specific cyclic aldehyde compound and an organic solvent. Patent Document 5 discloses a treatment method comprising the step of contacting a surface affected by a biofilm with a predetermined aqueous alkaline surfactant composition having a surfactant hydroxide molar concentration of 2-9. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Special Publication No. 2012-512199 [Patent Document 2] JP 2016-102227 A [Patent Document 3] JP 2016-035009 A [Patent Document 4] JP 2018-168097 A [Patent Document 5] Special Publication No. 2017-518432 Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention provides a method for removing biofilms that can effectively remove biofilms formed in aqueous systems such as devices and equipment that include components that come into contact with water, such as water cooling towers and bathtub piping. [Means for solving the problem]

[0007] The present invention relates to a method for removing a biofilm, which comprises contacting a metal-containing biofilm formed in an aqueous system with (a) a compound capable of generating hydroxyl radicals (hereinafter also referred to as component (a)) and (b) a reducing agent (hereinafter also referred to as component (b)). Effect of the Invention

[0008] According to the present invention, there is provided a method for removing a biofilm, which can effectively remove a biofilm formed in an aqueous system. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] Although the mechanism by which the biofilm removal method of the present invention exerts its effect has not been entirely elucidated, it is believed to be as follows. The present inventors have found that biofilms in aqueous systems can be decomposed or dispersed in water by hydroxyl radicals, thereby contributing to the removal of biofilms from equipment. In terms of the efficiency of action of hydroxyl radicals and biofilms formed on equipment, the presence of hydroxyl radicals on the surface of the biofilm is inefficient, and if hydroxyl radicals can be generated widely inside the biofilms formed on the equipment, a high biofilm removal efficiency can be expected. The components (a), (b), and even (c) of the present invention are all low molecular weights, and therefore tend to move widely inside the biofilms formed on the equipment. It has been found that, for example, metal ions originating from the equipment materials and the water supplied to the aqueous system are insoluble in water and accumulate in the biofilms generated in aqueous systems as salts, oxides, etc. The reducing agent (component (b)) approaches and reacts with these accumulated metal ions to become reduced metal ions (for example, Fe 3+ →Fe 2+). The reduced metal ions and the compound (component (a)) that is thought to be present in the vicinity of the reduced metal ions react with each other in a Fenton-like manner to generate hydroxyl radicals, which break the biofilm into pieces large enough to be dispersed in water. The metal ions oxidized by the Fenton-like reaction are further reduced by component (b), and the Fenton-like reaction with component (a) occurs continuously, so that the biofilm removal effect is sustained. In addition, when component (c) is used, an organic acid with a specific structure (component (c)) forms a complex with the metal ions, which makes it easier to make the insoluble metals present in the biofilm more water-soluble. As a result, the metal ions can be more uniformly present in the biofilm, and the reduction by the metal ions and component (b) is promoted, which also promotes the generation of hydroxyl radicals from component (a), which is thought to result in a higher biofilm removal effect. As described above, the biofilm removal method of the present invention utilizes trace amounts of metal ions accumulated in the biofilm, and therefore is believed to provide an efficient biofilm removal effect due to the generation of hydroxyl radicals that occur continuously in a wide range of the biofilm. As shown in the comparative example described below, even if metal ions are used together with component (a) and component (b) and contacted with a biofilm that does not contain metal ions, the biofilm cannot be removed. From this, it is presumed that in order to obtain the effects of the present invention, it is necessary for component (a) and component (b) to coexist and act in the presence of metal ions in the biofilm. The effects of the present invention are not limited to the above-mentioned mechanism of action.

[0010] [Component (a)] Component (a) is a compound capable of generating hydroxyl radicals. The component (a) may be a compound that generates hydroxyl radicals through a Fenton-like reaction. Examples of the component (a) include one or more compounds selected from hydrogen peroxide, percarbonates, and organic peracids, and from the viewpoints of availability, economy, and hydroxyl radical generating ability, one or more compounds selected from hydrogen peroxide and percarbonates are preferred. Examples of the percarbonates include alkali metal percarbonates such as sodium percarbonate and potassium percarbonate, and from the viewpoints of availability, economy, and hydroxyl radical generating ability, sodium percarbonate is preferred.

[0011] In the present invention, the term "Fenton-like reaction" can refer to the Fenton reaction and a reaction of hydrogen peroxide with a metal species other than iron ions to generate hydroxyl radicals (conveniently referred to as a pseudo-Fenton reaction). That is, in the present invention, the term "Fenton-like reaction" may be a concept including the Fenton reaction by iron ions and a reaction of hydrogen peroxide with a metal species other than iron ions to generate hydroxyl radicals similar to the Fenton reaction by metal species other than iron ions. The pseudo-Fenton reaction may be a chemical reaction in which hydroxyl radicals are generated from a compound that generates hydroxyl radicals using a metal ion other than iron ions as a catalyst. The Fenton reaction is a chemical reaction in which hydroxyl radicals are generated from hydrogen peroxide using divalent iron as a catalyst. On the other hand, the pseudo-Fenton reaction is a reaction in which, as a metal species, a metal species known to undergo a reaction similar to that with divalent iron, or a metal species selected from the transition elements of the fourth period of the periodic table discovered by the present invention (scandium, titanium, vanadium, chromium, manganese, cobalt, nickel, and copper), and, as a compound that generates hydroxyl radicals, a compound known to undergo a reaction similar to that with hydrogen peroxide with the metal species, or a compound selected from hydrogen peroxide, percarbonate, and organic peracid discovered by the present invention.

[0012] [(b) component] Component (b) is a reducing agent. Component (b) may be a compound having a reducing effect on a metal, such as iron. The metal on which component (b) of the present invention exerts a reducing effect refers to a metal in a state that can be reduced. (b) Ingredients include: (b-1) Compounds having an enediol structure: for example, ascorbic acid or a salt thereof, vitamin C extracted from natural products, tannic acid, erythorbic acid or a salt thereof, (b-2) Hydroxylamine: for example, N,N-diethylhydroxylamine, (b-3) Phenol-based reducing agents: for example, gallic acid, methylhydroquinone, dimethylhydroquinone, trimethylhydroquinone, t-butylhydroquinone, methoxyhydroquinone, chlorohydroquinone, (b-4) Other reducing agents: ascorbic acid derivatives or their salts, hydrosulfite, pyrosulfite, sulfite, hydrogen sulfite, thiosulfate, thiourea dioxide, Examples of the salts of these compounds include alkali metal salts such as sodium and potassium, and alkaline earth metal salts such as calcium salt. As component (b), from the viewpoints of easy availability and enhancing the biofilm removal effect (hereinafter also referred to as biofilm removal property), ascorbic acid or a salt thereof, or vitamin C extracted from a natural product is preferred, and ascorbic acid or a salt thereof is more preferred.

[0013] [(c) component] In the present invention, from the viewpoint of achieving a higher biofilm removal effect, it is preferable to contact the biofilm with (c) a monovalent or divalent organic acid or its salt (component (c)) having a first dissociation constant (hereinafter referred to as pKa1) of 1.2 to 4.6 inclusive, together with components (a) and (b). Among such organic acids or their salts, compounds having a reducing action are treated as component (b).

[0014] From the viewpoint of biofilm removing ability, the pKa1 of the component (c) is preferably 1.8 or more, more preferably 2.5 or more, and is preferably 3.9 or less, more preferably 3.3 or less.

[0015] The molecular weight of the organic acid of component (c), from the viewpoint of biofilm removing ability, may be, for example, 70 or more, and further 75 or more, further 95 or more, and 200 or less, further 180 or less, further 165 or less, and further 160 or less. From the viewpoint of biofilm removing ability, the organic acid of component (c) has a carbon number of preferably 2 or more, and preferably 8 or less, more preferably 6 or less, and further preferably 4 or less. From the viewpoint of biofilm removing ability, the organic acid of component (c) is preferably a carboxylic acid, and more preferably a monocarboxylic acid or dicarboxylic acid.

[0016] Examples of component (c) include one or more organic acids or salts thereof selected from hydroxymonocarboxylic acids having 2 or 3 carbon atoms and a molecular weight of 70 to 200, and further 180, dicarboxylic acids having 3 or 4 carbon atoms and a molecular weight of 70 to 200, and further 180, hydroxycarboxylic acids having 5 to 8 carbon atoms and a molecular weight of 70 to 200, and further salts thereof.

[0017] Examples of hydroxymonocarboxylic acids having 2 or 3 carbon atoms and a molecular weight of 70 to 200 include glycolic acid (pKa1: 3.83, Mw: 76.1), lactic acid (pKa1: 3.86, Mw: 90.1), and 3-hydroxypropionic acid (pKa1: 4.5, Mw: 90.1).

[0018] Examples of dicarboxylic acids having 3 or 4 carbon atoms and a molecular weight of 70 to 200 include tartaric acid (pKa1:2.98, Mw:150.1), fumaric acid (pKa1:3.02, Mw:116.1), maleic acid (pKa1:1.92, Mw:116.1), malic acid (pKa1:3.4, Mw:134.1), succinic acid (pKa1:4.19, Mw:118.1), and malonic acid (pKa1:2.9, Mw:104.1).

[0019] An example of a hydroxycarboxylic acid having 5 to 8 carbon atoms and a molecular weight of 70 to 200 is gluconic acid (pKa1: 3.86, Mw: 196).

[0020] From the viewpoint of biofilm removal ability, the (c) component is preferably one or more organic acids or salts thereof selected from malonic acid, malic acid, 3-hydroxypropionic acid, succinic acid, lactic acid, tartaric acid, glycolic acid, maleic acid, fumaric acid, gluconic acid, and salts thereof. From the viewpoint of biofilm removing ability, the component (c) is more preferably one or more organic acids or salts thereof selected from tartaric acid, fumaric acid, maleic acid, gluconic acid, and salts thereof.

[0021] Examples of the organic acid salt of component (c) include alkali metal salts such as sodium and potassium, and alkaline earth metal salts such as calcium.

[0022] [Method for removing biofilm] In the biofilm removal method of the present invention, components (a) and (b), and optionally further component (c), are contacted with a metal-containing biofilm formed in an aqueous system. In the present invention, it is preferable to contact the biofilm with a liquid composition containing components (a), (b), and water. It is preferable that the liquid composition further contains component (c).

[0023] In the liquid composition, the concentration of component (a) can be set by selecting and combining the upper and lower limit values ​​from the viewpoint of biofilm removal ability, for example, 50 ppm or more, further 60 ppm or more, further 80 ppm or more, and 50,000 ppm or less, further 30,000 ppm or less, further 10,000 ppm or less, further 5,000 ppm or less, further 3,000 ppm or less, further 1,000 ppm or less, further 600 ppm or less, and further 500 ppm or less. In the liquid composition, the concentration of component (b) can be set by selecting and combining the upper and lower limit values ​​from the viewpoint of biofilm removal ability, for example, 50 ppm or more, further 60 ppm or more, further 80 ppm or more, and 5000 ppm or less, further 3000 ppm or less, further 1000 ppm or less, further 600 ppm or less, and further 500 ppm or less. When component (c) is used, the concentration of component (c) in the liquid composition can be set by selecting and combining the upper and lower limit values ​​from the viewpoint of biofilm removal ability, for example, 50 ppm or more, further 60 ppm or more, further 80 ppm or more, and 5000 ppm or less, further 3000 ppm or less, further 1000 ppm or less, further 600 ppm or less, and further 500 ppm or less.

[0024] In the present invention, from the viewpoint of biofilm removal ability, it is preferable to contact component (a) with component (b) in a mass ratio of component (a) / component (b) of 0.01 or more, preferably 0.1 or more, and 100 or less, preferably 10 or less, preferably 9.5 or less, preferably 4.5 or less, preferably 1.5 or less, and more preferably 0.5 or less. When the liquid composition is used, from the viewpoint of biofilm removal ability, the mass ratio (a) / (b) of the concentration of component (a) to the concentration of component (b) in the composition is preferably 0.01 or more, more preferably 0.1 or more, and preferably 100 or less, more preferably 10 or less, even more preferably 9.5 or less, still more preferably 4.5 or less, still more preferably 1.5 or less, and even more preferably 0.5 or less.

[0025] In the present invention, from the viewpoint of biofilm removal ability, it is preferable to contact component (a) with component (c) in a mass ratio of component (a) / component (c) of 0.01 or more, preferably 0.1 or more, and 100 or less, more preferably 10 or less. When the liquid composition is used, the mass ratio (a) / (c) of the concentration of component (a) to the concentration of component (c) in the composition is preferably 0.01 or more, more preferably 0.1 or more, and is preferably 100 or less, more preferably 10 or less, from the viewpoint of biofilm removal ability.

[0026] In the present invention, from the viewpoint of biofilm removal ability, it is preferable to contact the (b) component with the (c) component in a mass ratio of the (b) component / the (c) component of 0.01 or more, preferably 0.1 or more, and 100 or less, more preferably 10 or less. When the liquid composition is used, the mass ratio (b) / (c) of the concentration of component (b) to the concentration of component (c) in the composition is, from the viewpoint of biofilm removal ability, preferably 0.01 or more, more preferably 0.1 or more, and preferably 100 or less, more preferably 10 or less.

[0027] In the present invention, even a small amount of surfactant contacted with a biofilm shows an excellent biofilm removal effect. From the viewpoint of equipment management that does not cause problems such as cavitation due to foaming, economic efficiency, and biofilm removal, the liquid composition may have a surfactant concentration of, for example, less than 100 ppm, further 50 ppm or less, further 10 ppm or less, or further 0 ppm. The liquid composition may be substantially free of surfactant. Here, "substantially free of surfactant" may mean that the surfactant concentration is within the above range.

[0028] The liquid composition may be free or substantially free of metal ions involved in the Fenton-like reaction, specifically, ions of metals selected from iron, scandium, titanium, vanadium, chromium, manganese, cobalt, nickel, and copper. Here, "substantially free of metal ions" may mean that even if the metal ions are contained, the amount is extremely small, and the Fenton-like reaction does not occur sufficiently.

[0029] In the present invention, the contact of component (a) and component (b) can be carried out at a pH of 2.0 or more, further 2.5 or more, further 3.0 or more, and further 3.5 or more from the viewpoint of preventing damage to equipment, and at a pH of 10.0 or less, further 8.0 or less, further 7.5 or less, and further 6.5 or less from the viewpoint of the biofilm removal effect. That is, the present invention may be a method of contacting component (a) and component (b) with a biofilm under conditions of a pH of 2.0 or more, further 2.5 or more, further 3.0 or more, and 10.0 or less, further 8.0 or less, further 7.5 or less, and further 6.5 or less. When the liquid composition is used, from the same viewpoint, the pH of the composition may be 2.0 or more, further 2.5 or more, further 3.0 or more, further 3.5 or more, and 10.0 or less, further 8.0 or less, further 7.5 or less, further 6.5 or less.

[0030] The biofilm formed in the aqueous system and removed by the present invention contains a metal in the formed biofilm. The metal contained in the biofilm is preferably one that reacts with the (a) and (b) components to generate hydroxyl radicals. Such metals are not particularly limited, but include one or more metals selected from transition elements in the fourth period of the periodic table, such as scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, and copper. From the viewpoint of efficiently generating hydroxyl radicals, one or more metals selected from iron, manganese, nickel, and copper are preferred. Tap water, well water, etc. are used as water that flows, circulates, and stays in the aqueous system, and microorganisms such as Pseudomonas aeruginosa are contained in the water, but at the same time, there are metal ions dissolved in small amounts from the natural world or from equipment materials such as pipes and tanks made of metal that constitute the aqueous system. These microorganisms form a biofilm together with the water in which the metal is dissolved and compounds such as sugars and proteins produced by the microorganisms, and it is considered that the metal is contained in the biofilm because the formed biofilm further takes in new metals from the water in the system. Metals are thought to exist as ions in water, but in biofilms they are thought to form salts or oxides with compounds excreted by microorganisms and accumulate in the biofilm.

[0031] From the viewpoint of availability in selecting materials for piping installation, many of the constituent materials of aqueous systems contain metals such as iron, and metals from the constituent materials may be incorporated into biofilms formed in aqueous systems. Therefore, the biofilms that are the subject of the present invention may contain iron.

[0032] The biofilm to be treated by the present invention contains, from the viewpoint of biofilm removability, preferably 0.5 ng or more, more preferably 1.5 ng or more, and even more preferably 5 ng or more of metal per 1 mg of wet biofilm, and the upper limit is not particularly limited as long as it is an amount that can be accumulated in the biofilm, but from the viewpoint of efficiently proceeding the reaction without causing an excessively rapid Fenton-like reaction, it contains preferably 50,000 ng or less, more preferably 20,000 ng or less, and even more preferably 10,000 ng or less. The amount of metal in the biofilm can be measured by the method described in the Examples below.

[0033] The aqueous system of the present invention is not particularly limited as long as it is a system in which contact with an aqueous liquid occurs, but may be, for example, a system that functions by the circulation or storage of an aqueous liquid, and may be a device composed of a member that comes into contact with water, such as a pipe, a tank, or a pool, and a facility including the device. The water may be water itself, or an aqueous medium containing water and other substances. As an example, the aqueous system is a cooling system equipped with a water-cooled water cooling tower. As another example, the aqueous system may include a hot water piping section selected from a water distribution path such as a water distribution pipe, for example, a hot water piping section of a boiler, and a hot water piping section of a circulating hot water supply pipe to which hot water is supplied from a hot water tank. The aqueous system may also include a water storage tank. Furthermore, other examples of the aqueous system include industrial cooling pools, industrial water supply, storage or drainage paths, drainage treatment facilities, running water heating systems, tanks, pools, baths, filtration facilities, papermaking facilities in paper factories, aquarium tanks and water circulation paths, ultrapure water devices, aquaculture facilities, and plant factories.

[0034] The present invention relates to removing metal-containing biofilms formed in aqueous systems, particularly at sites of contact with water in aqueous systems. The aqueous system of the present invention is preferably one equipped with a mechanism for contacting with water, such as a mechanism for periodically circulating water or a mechanism for circulating water. In this case, by adding component (a), component (b), and optionally component (c) of the present invention to the water to be contacted and operating the aqueous system to contact the water, component (a), component (b), and optionally component (c) can come into contact with the biofilm and remove the biofilm that has developed in the aqueous system.

[0035] The time for which the components (a) and (b) are contacted with the biofilm is, for example, 3 minutes or more, further 5 minutes or more, further 10 minutes or more, further 30 minutes or more, and 48 hours or less, further 30 hours or less, from the viewpoint of workability and simplicity in facility management. When the liquid composition is used, the contact time between the liquid composition and the biofilm may be within this range.

[0036] The temperature at which the components (a) and (b) are brought into contact with the biofilm is, for example, 5° C. or higher, further 10° C. or higher, and 85° C. or lower, further 60° C. or lower. When the liquid composition is used, the temperature of the liquid composition may be within this range.

[0037] When applying the (a), (b), and (c) components of the present invention to an aqueous system, a composition containing these components at a concentration suitable for contact may be used, or a composition containing these components at a high concentration may be prepared and diluted with water. Alternatively, the (a), (b), and (c) components may be used individually, or as a premix or predissolved solution, dissolved in water flowing, circulating, or remaining in the aqueous system. The pH may be adjusted as necessary. The (a), (b), and (c) components may be supplied in the form of a liquid, solid, or aqueous solution, depending on the compound. When using the liquid composition, it is preferable to prepare the composition immediately before applying it to the aqueous system from the viewpoint of the liquid stability of the composition. It is also preferable to dissolve the (a), (b), and (c) components of the present invention in water flowing, circulating, or remaining in the aqueous system, and contact these components with the biofilm.

[0038] In the method for removing a biofilm of the present invention, a composition containing one or more metals, preferably one selected from iron, manganese, nickel and copper, may be contacted with a biofilm formed in an aqueous system to form a biofilm containing a metal or a biofilm with an increased content of metal, and then the component (a) and the component (b) may be contacted. Specifically, a composition containing one or more metals selected from iron, manganese, nickel and copper, and an aqueous composition containing the metal at a concentration of, for example, 1 ppm to 100 ppm, may be contacted with a biofilm formed in an aqueous system for a predetermined period of time, for example, 3 minutes or more, preferably 5 minutes or more, more preferably 10 minutes or more, more preferably 30 minutes or more, even more preferably 1 hour or more, even more preferably 2 hours or more, and from the viewpoint of workability, for example, for a period of 3 days or less, for example, by circulating, circulating or retaining the composition in the aqueous system, and then, if necessary, the composition may be replaced with water containing only a very small amount of metal, such as ordinary tap water or well water, and the composition may be circulated, circulated or retained in the aqueous system for a predetermined period of time, and then the component (a) and the component (b) may be contacted. It is believed that by contacting the biofilm with a composition containing metal ions at a higher concentration than ordinary tap water, well water, etc., prior to contacting component (a) with component (b), more metals will accumulate in the biofilm, thereby further improving the biofilm removal effect.

[0039] In addition, even if the metal is contacted at the same time when the component (a) and the component (b) are contacted with the biofilm, the effect of the present invention is not manifested. In this case, even if the concentration of the metal is made higher than the concentration contained in normal tap water or well water, the effect of the present invention is not manifested. This is thought to be because the Fenton-like reaction occurs explosively at once in the water, and the biofilm cannot be acted on and decomposed, and the biofilm removal effect cannot be manifested. Therefore, in the present invention, it is presumed that the excellent effect is exhibited by the Fenton-like reaction occurring inside the biofilm, and if water is used when the component (a) and the component (b) are contacted with the biofilm, it is desirable to reduce the metal ion concentration involved in the Fenton-like reaction as much as possible in the water.

[0040] In the method of the present invention, for example, the liquid composition is dissolved in water flowing, circulating or stagnant in a water-based system and contacted with a biofilm. At that time, an external force may be applied. In the case of a portion that cannot be directly touched by hands, such as the inside of a drainage pipe, the liquid composition can be brought into contact with the biofilm by flowing or circulating, etc., to sufficiently remove the biofilm. On the other hand, in the case of a portion that can be directly touched by hands, such as a pool or a bath, the biofilm can be removed by simply dissolving the liquid composition in stagnant water, etc., and contacting the biofilm, but the removal of the biofilm can be further ensured by applying a light external force such as rubbing with a mop or a rag. EXAMPLES

[0041] <Example 1 and Comparative Example 1> Liquid compositions to be contacted with aqueous systems were prepared using the components shown in Table 1, and the following tests were carried out. The results are shown in Table 1. The pH of the compositions in Table 1 was adjusted with 10 mM acetate buffer (to adjust pH to 4.0) or 10 mM phosphate buffer (to adjust pH to 7.0), and then each component was adjusted to a specified concentration. The remainder of the compositions in Table 1 was water.

[0042] [Biofilm removal test (water cooling tower model)] (1) Preparation of biofilm To create the biofilm, we used an aqueous system model using an annular reactor (manufactured by Art Science Co., Ltd.) with a 100 mL culture tank. The culture tank of the annular reactor was equipped with a cylindrical rotor that rotated at a speed of 160 revolutions per minute, and a test piece (SUS304) was attached to this rotor in advance. Cooling water collected from a water-cooled water cooling tower for cooling the reaction tank equipment of a chemical plant was supplied to the annular reactor culture tank (maintained at 30°C), and the culture was continued for 3 weeks to form a biofilm on the test piece.

[0043] (2) Measurement of metal content in biofilms A sample amount of biofilm was taken from the biofilm formed in (1) in a wet state by removing the surface moisture with filter paper, and metals were extracted with phosphate-buffered saline. The amount of metals was measured by ICP-MS, and the amount of metals contained in 1 mg of biofilm in a wet state was calculated. As a result, it was confirmed that the biofilm contained 68 ng / mg Fe, 4.6 ng / mg Ni, and 3.7 ng / mg Cu.

[0044] (3) Biofilm removal test A test piece on which a biofilm had formed was immersed in each well of a 6-well plate to which 5 mL of a liquid composition containing the components in Table 1 at the concentrations in Table 1 or a control (ultrapure water) had been added, and the plate was shaken at 30°C and 60 rpm for 16 hours. Each test piece was stained with 0.1% crystal violet, washed with ultrapure water, and the staining solution was extracted with 1 mL of ethanol, and the absorbance (OD570) was measured. Based on the measured OD570, the biofilm removal rate was measured according to the following formula. The blank refers to the OD570 of ethanol. Biofilm removal rate (%) = {(control OD570 - blank OD570) - (OD570 of the example or comparative example - blank OD570)} x 100 / (control OD570 - blank OD570) In addition, the higher the biofilm removal rate in this test, the greater the effect; a 5% difference in the biofilm removal rate in this test can be recognized as a significant difference, and a 10% difference can be recognized as an even clearer difference.

[0045] [Table 1]

[0046] When removing biofilms using external force, for example, when removing biofilms by scrubbing with a mop, rag, etc., a biofilm removal rate of 33% or more according to this evaluation is sufficient to achieve the desired effect. When the external force is smaller, for example, when removing biofilms by distribution or circulation of the composition in Table 1, a biofilm removal rate of 40% or more is sufficient to remove the biofilm, and is therefore more preferable.

[0047] <Example 2 and Comparative Example 2> Liquid compositions to be brought into contact with an aqueous system were prepared using the components shown in Table 2, and the following tests were carried out. The results are shown in Table 2. The pH of the compositions in Table 2 was adjusted with 10 mM acetate buffer or 10 mM phosphate buffer, and then each component was adjusted to a specified concentration. The remainder of the compositions in Table 2 was water.

[0048] [Biofilm removal test (bathtub piping model)] (1) Preparation of biofilm To prepare the biofilm, an aqueous system model was used using an annular reactor (manufactured by Art Science Co., Ltd.) with a 100 mL culture tank. A cylindrical rotor rotating at a speed of 160 revolutions per minute was installed in the culture tank of the annular reactor, and a test piece (SUS304) was attached to this rotor in advance. Bath water collected from a circulating bath was supplied to the annular reactor culture tank (maintained at 40°C), while R2A medium was added to the supply water as a dirt load treatment so that the concentration increased by 10 ppm every day, and the biofilm was formed on the test piece by culturing for one week.

[0049] (2) Measurement of metal content in biofilms The amount of metal contained in the biofilm formed in (1) was calculated in the same manner as in Example 1. As a result, it was confirmed that the biofilm contained 220 ng / mg Fe and 6.8 ng / mg Mn.

[0050] (3) Biofilm removal test A test piece on which a biofilm had formed was immersed in each well of a 6-well plate to which 5 mL of a liquid composition containing the components in Table 2 at the concentrations in Table 2 or a control (ultrapure water) had been added, and the plate was shaken at 40°C and 60 rpm for 30 minutes. Each test piece was stained with 0.1% crystal violet, washed with ultrapure water, and the staining solution was extracted with 1 mL of ethanol, and the absorbance (OD570) was measured. Based on the measured OD570, the biofilm removal rate was measured according to the following formula. The blank refers to the OD570 of ethanol. Biofilm removal rate (%) = {(control OD570 - blank OD570) - (OD570 of the example or comparative example - blank OD570)} x 100 / (control OD570 - blank OD570)

[0051] [Table 2]

[0052] <Example 3 and Comparative Example 3> The biofilm removal rate was measured in the same manner as in Example 1 using the test pieces on which the biofilms were formed as prepared in Examples 1 and 2. Furthermore, the biofilm removal rate for biofilms caused by Pseudomonas aeruginosa or biofilms caused by bacteria of the Sphingomonas family was measured by the method described below. The results are shown in Table 3.

[0053] (1) Preparation of biofilm (Pseudomonas aeruginosa) Pseudomonas aeruginosa PAO1 strain (ATCC 15692) was cultured in R2A medium at 30°C for 16 hours with shaking. The resulting culture solution was diluted 1 / 100 with R2A medium, and 200 μL was added to each well of a 96-well microplate and cultured at 30°C for 3 days to create a biofilm.

[0054] (2) Preparation of biofilm (Sphingomonasaceae bacteria) Sphingomonas bacteria isolated from cooling tower cooling water were cultured in R2A medium at 30°C for 16 hours with shaking. The resulting culture solution was diluted 1 / 100 with R2A medium containing 0.1μM iron chloride (III), 100μM manganese chloride (II), 100μM nickel chloride (II), or 100μM copper chloride (II), and 200μL of the diluted solution was added to each well of a 96-well microplate and cultured at 30°C for 3 days to produce a biofilm.

[0055] (3) Measurement of metal content in biofilms The amounts of metals contained in the biofilms formed in (1) and (2) were calculated in the same manner as in Example 1. As a result, it was confirmed that the biofilm (1) did not contain any metals, while the biofilm (2) contained 19.8 ng / mg Fe, 7.8 ng / mg Mn, 1.8 ng / mg Ni, and 12.9 ng / mg Cu, respectively.

[0056] (4) Biofilm removal test The culture medium in the wells was replaced with a liquid composition containing the components in Table 3 at the concentrations in Table 3 or a control (ultrapure water), and allowed to act at 30°C for 16 hours. Thereafter, the liquid composition or ultrapure water was discarded, and each well was stained with 0.1% crystal violet, washed twice with ultrapure water, 200 μL of ethanol was added, and the absorbance (OD570) was measured. Based on the measured OD570, the biofilm removal rate was measured according to the following formula. The blank refers to the OD570 of ethanol. Biofilm removal rate (%) = {(control OD570 - blank OD570) - (OD570 of the example or comparative example - blank OD570)} x 100 / (control OD570 - blank OD570)

[0057] [Table 3]

[0058] As shown in Examples 3-1, 3-2, and 3-3 in Table 3, when a liquid composition containing components (a) and (b) is brought into contact with a biofilm containing a metal, the biofilm removal rate is very high, and the biofilm can be removed satisfactorily. On the other hand, as shown in Comparative Example 3-1, it is understood that the biofilm cannot be removed even when the liquid composition is brought into contact with a biofilm not containing a metal. Moreover, it is understood from Comparative Example 3-2 that the biofilm cannot be removed even when a metal (Fe) is present in the liquid composition and brought into contact with a biofilm not containing a metal.

[0059] <Example 4> A liquid composition to be contacted with an aqueous system was prepared using the components shown in Table 4, and the following tests were carried out. The results are shown in Table 4.

[0060] (1) Preparation of biofilm (Sphingomonasaceae bacteria) The biofilm was produced by the method of producing a biofilm in Example 3(2) without adding any metal.

[0061] (2) Measurement of metal amounts in biofilms The amount of metal contained in the biofilm formed in (1) was calculated in the same manner as in Example 1. As a result, the biofilm (1) did not contain any metals.

[0062] (3) Biofilm removal test The culture solution in the well was replaced with an iron (III) chloride solution dissolved in ultrapure water to a concentration of 1.6 ppm, and the solution was left to stand for 1, 5, 10, 30, or 120 minutes, after which the solution was replaced with phosphate buffered saline to wash away the remaining iron (III) chloride solution. The liquid composition in Table 4 was then prepared with 10 mM phosphate buffer, and a biofilm removal test was carried out in the same manner as in Example 3 to measure the removal rate.

[0063] [Table 4]

[0064] <Example 5> A biofilm removal test was carried out in the same manner as in Example 2, using the test piece on which the biofilm was formed as in Example 2 and a liquid composition to be brought into contact with the aqueous system prepared using the components shown in Table 5. However, the remainder of the liquid composition was ultrapure water, and the cleaning time was 60 minutes. The results are shown in Table 5.

[0065] [Table 5]

Claims

1. A method for removing a biofilm, comprising contacting a metal-containing biofilm formed in an aqueous system with a liquid composition containing (a) a compound capable of generating hydroxyl radicals (hereinafter also referred to as component (a)), (b) a reducing agent (hereinafter also referred to as component (b)), and (c) a monovalent or divalent organic acid or a salt thereof having a first dissociation constant of 1.2 or more and 4.6 or less (hereinafter also referred to as component (c)), and water, (b) component is ascorbic acid or a salt thereof; (c) The component is one or more organic acids or salts thereof selected from tartaric acid, fumaric acid, maleic acid, gluconic acid, and salts thereof; How to remove biofilm.

2. The method for removing a biofilm according to claim 1, wherein in the liquid composition, the concentration of component (a) is 50 ppm or more and 5,000 ppm or less, the concentration of component (b) is 50 ppm or more and 50,000 ppm or less, and the concentration of component (c) is 50 ppm or more and 5,000 ppm or less.

3. The method for removing a biofilm according to claim 1 or 2, wherein in the liquid composition, the concentration of component (a) is 50 ppm or more and 5,000 ppm or less, the concentration of component (b) is 50 ppm or more and 5,000 ppm or less, and the concentration of component (c) is 50 ppm or more and 5,000 ppm or less.

4. A method for removing a biofilm described in any one of claims 1 to 3, wherein contact with the liquid composition is performed at a pH of 2.0 or higher and 10.0 or lower.

5. The method for removing a biofilm according to any one of claims 1 to 4, wherein the component (a) is one or more selected from hydrogen peroxide, percarbonate, and organic peracid.

6. A method for removing a biofilm described in any one of claims 1 to 5, wherein the mass ratio (a) / (b) of the concentration of component (a) to the concentration of component (b) in the liquid composition is 0.01 or more and 100 or less.

7. A method for removing a biofilm described in any one of claims 1 to 6, wherein the mass ratio (a) / (c) of the concentration of component (a) to the concentration of component (c) in the liquid composition is 0.01 or more and 100 or less.

8. The method for removing a biofilm according to any one of claims 1 to 7, wherein the biofilm contains one or more metals selected from iron, manganese, nickel and copper.

9. The method for removing a biofilm according to any one of claims 1 to 8, wherein the aqueous system is a cooling system equipped with a water-cooled water cooling tower.

10. The method for removing a biofilm according to any one of claims 1 to 9, wherein the aqueous system includes a water distribution channel and / or a water storage tank.

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