Method for producing a bacterial admixture for addition to cement compositions

Abstract

The present invention provides a method for producing a bacteria-containing admixture (for addition to cement compositions) for producing an admixture for addition to cement compositions that does not significantly affect the fresh properties of concrete when using a culture solution in which live bacteria have been grown in concrete. The present invention also provides a method for producing a bacteria-containing admixture (for addition to cement compositions) that solves the problems of the culture solution in which live bacteria have been grown having an excessively strong odor, the culture solution being prone to spoilage and resulting in a short shelf life, and the difficulty of storing a large amount of culture solution due to its volume. [Solution] A method for producing a bacteria-containing admixture for addition to a cement composition includes a spore-forming bacteria growth step in which aerobic spore-forming bacteria are cultured and grown; a solid-liquid separation step in which the culture solution in which the aerobic spore-forming bacteria have been grown is separated into a solid fraction containing the spore-forming bacteria and a liquid fraction containing the culture metabolic solution; and a mixing step in which the solid fraction containing the spore-forming bacteria is mixed with any admixture to obtain a bacteria-containing admixture.

Description

[Technical Field]

[0001] The present invention relates to a method for producing a bacteria-containing admixture for addition to a cement composition. [Background technology]

[0002] Hardened cement products such as concrete and mortar are widely used for construction purposes, and in particular, reinforced concrete in which steel materials such as reinforcing bars are embedded in concrete is widely used as building structures.

[0003] Calcium hydroxide is produced during the hardening process of concrete and mortar. This calcium hydroxide creates a high pH environment, which causes a passive film (oxide compound film) to form on the steel in reinforced concrete, placing the steel in an environment that is less susceptible to corrosion.

[0004] It is known that as concrete deteriorates over time (for example, when cracks appear on the surface of the concrete), calcium hydroxide in the concrete dissolves in water that penetrates through the cracks and then reacts with carbon dioxide in the air to precipitate calcium carbonate, resulting in the loss of calcium hydroxide. This loss of calcium hydroxide causes the concrete's pH to shift from alkaline to acidic, a phenomenon known as neutralization. This neutralization and the penetration of chloride ions destroy the passivation film (formed by calcium hydroxide) around steel embedded in the concrete, causing the steel to corrode.

[0005] To prevent this corrosion of steel materials, for example, a method has been proposed in which microbial metabolism is utilized to reduce dissolved oxygen in concrete, thereby preventing corrosion of reinforcing bars (Patent Document 1).In prior art methods such as Patent Document 1, live bacteria are grown in a culture solution by shaking culture of a bacterial strain, and the culture solution in which the live bacteria have grown is then poured into the concrete. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent No. 6716331 Summary of the Invention [Problem to be solved by the invention]

[0007] However, the conventional methods described in Patent Document 1 and the like have the drawback that if a large amount of culture solution is added to the concrete, it significantly affects the fresh properties of the concrete (particularly the slump and air content). In particular, an increase in the air content has a negative effect on the performance of the concrete after hardening, so in order to suppress this increase in the air content, it has been necessary to add a large amount of an antifoaming agent, which is not normally used.

[0008] Furthermore, when using the culture medium itself in which live bacteria have been propagated, there are problems such as the odor of the culture medium being too strong, the culture medium being easily spoiled and therefore having a short shelf life, and the difficulty of storing a large amount of the culture medium due to its volume. [Means for solving the problem]

[0009] The present inventors have conducted extensive research to achieve the above object, and as a result have found that the above problems can be solved by separating a culture medium in which aerobic spore-forming bacteria have been grown into a solid fraction containing the spore-forming bacteria and a liquid fraction containing a culture metabolic solution, and using the solid fraction containing the spore-forming bacteria in a cement composition, thereby completing the present invention.

[0010] To achieve the above object, the method for producing a bacteria-containing admixture for addition to a cement composition according to the invention described in claim 1 comprises: a step of culturing and growing aerobic spore-forming bacteria; a solid-liquid separation step of separating the culture solution in which the aerobic spore-forming bacteria have been grown into a solid fraction containing the spore-forming bacteria and a liquid fraction containing a culture metabolic solution; The method is characterized by including a mixing step of mixing the solid fraction containing the spore-forming bacteria with any admixture to obtain a bacteria-containing admixture.

[0011] The invention described in claim 2 is the invention described in claim 1, After the solid-liquid separation step, the solid fraction containing the spore-forming bacteria is added to a new medium, and a medium exchange step is performed to obtain a mixed solution of the solid fraction and the new medium. The mixing step is characterized in that, instead of the solid fraction containing the spore-forming bacteria, a mixture of the solid fraction and a new medium is mixed with any admixture to obtain a bacteria-containing admixture.

[0012] The invention described in claim 3 is the invention described in claim 1 or 2, The method is characterized in that it comprises a refrigerated storage step of storing the solid fraction or a mixture of the solid fraction and a new medium in a refrigerator after the solid-liquid separation step. [Effects of the Invention]

[0013] The method for producing a bacteria-containing admixture for addition to cement compositions of the present invention can produce a bacteria-containing admixture that does not significantly affect the fresh properties of concrete when a culture solution in which live bacteria have been grown is used for concrete. Furthermore, the method for producing a bacteria-containing admixture of the present invention can solve the problems of the culture solution in which live bacteria have been grown having an excessively strong odor, the problem of the culture solution having a short shelf life due to its tendency to spoil, and the problem of the culture solution's large volume making it difficult to store in large quantities. DETAILED DESCRIPTION OF THE INVENTION

[0014] The method for producing a bacteria-containing admixture of the present invention includes a spore-forming bacterium growth step in which aerobic spore-forming bacteria are cultured and grown, a solid-liquid separation step in which the culture solution in which the aerobic spore-forming bacteria have been grown is separated into a solid fraction containing the spore-forming bacteria and a liquid fraction containing a culture metabolic solution, and a mixing step in which the solid fraction containing the spore-forming bacteria is mixed with any admixture to obtain a bacteria-containing admixture. Note that this bacteria-containing admixture is to be added to a cement composition.

[0015] <Spore-forming bacteria growth process in which aerobic spore-forming bacteria are cultured and grown> [Aerobic spore-forming bacteria] The aerobic spore-forming bacteria are not particularly limited as long as they are bacteria that consume oxygen, generate carbon dioxide, and have the ability to form spores (spores, microspores).

[0016] As the spore-forming bacteria, for example, one or more species of Bacillus subtilis (genus Bacillus), actinomycetes (genus Streptomyces, etc.), fungi (phyla Imperfectomycota, Ascomycota, Zygomycota, Basidiomycota, Chytridiomycota), etc. can be used. Among these, aerobic bacteria that produce endospores, such as Bacillus subtilis, are preferred.

[0017] The枯草 bacterium is not particularly limited. For example, Bacillus altitudinis, Bacillus subtilis, Bacillus subtilis var. natto (natto bacterium), Bacillus amyloliquefaciens, Bacillus pumils, Bacillus lentus, Bacillus laterosporus, Bacillus alvei, Bacillus popilliae, Bacillus licheniformis, Bacillus coagulans, Bacillus cereus, Bacillus halodurans, Bacillus acidicola, Bacillus acidopullulyticus, Bacillus acidovorans, Bacillus aeolius, Bacillus aestuarii, Bacillus garadhaerens, Bacillus akibai, Bacillus alcaliinulinus, Bacillus alcalophilus, Bacillus algicola, Bacillus alkalitolerans, Bacillus alkalogaya, Bacillus alveayuensis, Bacillus amiliensis, Bacillus aminovorans, Bacillus aquimaris, Bacillus arbutinivorans, Bacillus arenosi, Bacillus arseniciselenatis, Bacillus arsenicus, Bacillus arvi, Bacillus asahii, Bacillus atrophaeus, Bacillus axarquiensis, Bacillus azotoformans, Bacillus badius, Bacillus baekryungensis, Bacillus barbaricus, Bacillus bataviensis, Bacillus benzoevorans, Bacillus bogoriensis, Bacillus borophilicus, Bacillus borotolerans, Bacillus caldolyticus, Bacillus It should be noted that the original text seems to be incomplete. There is an unfinished "Bacillus" at the end. If you can provide the complete text, a more accurate translation can be obtained.One or more species can be selected from the group consisting of Bacillus caldotenax, Bacillus caldovelox, Bacillus carboniphilus, Bacillus casamancensis, Bacillus catenulatus, Bacillus cellulosilyticus, Bacillus sphericus, Bacillus thuringiensis, Bacillus clausii, etc.

[0018] As Bacillus altitudinis, one or more species such as Bacillus altitudinis AH1 strain (accession number NITE P-03987 (National Institute of Technology and Evaluation Patent Microorganisms Depositary Center)) and Bacillus altitudinis AH2 strain (accession number NITE P-03988 (National Institute of Technology and Evaluation Patent Microorganisms Depositary Center)) can be used. These spore-forming bacteria may be either self-cultured or commercially available.

[0019] [Culture conditions for aerobic spore-forming bacteria] The culture conditions for aerobic spore-forming bacteria (e.g., type of medium, culture temperature, shaking speed, culture time, and pH in the medium) are not particularly limited as long as they are normal culture conditions for spore-forming bacteria. The culture conditions for pre-culture and main culture are, for example, as follows.

[0020] (preculture) Examples of the culture medium include LB medium and yeast extract medium, with LB medium (Miller) being preferred. The culture temperature is, for example, in the range of about 25 to 35°C, and preferably about 30°C. The shaking speed is, for example, in the range of about 50 to 150 rpm, and preferably about 100 rpm. The culture time is, for example, in the range of about 12 to 48 hours, and preferably about 24 hours. The pH of the culture medium is, for example, in the range of about 7 to 10, and preferably about 8.

[0021] (Main culture) Examples of the medium include LB medium and yeast extract medium, with yeast extract medium being preferred. The culture temperature is, for example, in the range of about 25 to 35°C, and preferably about 30°C. The shaking speed is, for example, in the range of about 50 to 150 rpm, and preferably about 100 rpm. The culture time is, for example, in the range of about 12 to 48 hours, and preferably about 24 hours. The pH of the medium is, for example, in the range of about 7 to 10, and preferably about 8.

[0022] <Solid-liquid separation step in which a culture medium in which aerobic spore-forming bacteria have been grown is separated into a solid fraction containing spore-forming bacteria and a liquid fraction containing culture metabolic liquid> A culture medium in which aerobic spore-forming bacteria have been grown is separated into a solid fraction containing the spore-forming bacteria and a liquid fraction containing the culture metabolic solution (fermentation metabolites (waste products such as organic acids and carbon dioxide) generated by microbial metabolism during cultivation). The means for solid-liquid separation is not limited as long as it can separate the solid fraction containing the spore-forming bacteria from the liquid fraction containing the culture metabolic solution. Examples of the means include suction filtration and centrifugation, with suction filtration being preferred.

[0023] Examples of suction filtration include suction filtration using a known membrane filter unit. The pore size of the membrane filter can be selected appropriately depending on the type of spore-forming bacteria, and is preferably in the range of 0.1 μm to 0.5 μm, particularly 0.2 μm. The type of membrane filter can be selected appropriately depending on the type of spore-forming bacteria, and is preferably a PES membrane filter or Teflon (registered trademark), particularly a PES membrane filter. In the case of suction filtration using a membrane filter unit, a solid fraction containing spore-forming bacteria remains on the membrane filter, and a liquid fraction containing the culture metabolite solution passes through the membrane filter.

[0024] The solid-liquid separation process removes the culture metabolic liquid from the culture medium in which spore-forming bacteria are grown (i.e., fermentation metabolites (waste products such as organic acids and carbon dioxide) generated by microbial metabolism during cultivation are removed), eliminating the problem of the culture medium (or the admixture containing the solid fraction containing spore-forming bacteria) having an excessively strong odor.

[0025] In addition, because the culture metabolic liquid, which occupies a large portion of the volume of the culture medium, has been removed (i.e., the volume of the culture medium has been reduced), it has become possible to stockpile a large amount of the solid fraction containing spore-forming bacteria (or an admixture containing said solid fraction).Furthermore, because the volume of the culture medium has been reduced, the solid fraction containing spore-forming bacteria (or an admixture containing said solid fraction) can be easily stored in a refrigerator, etc., which solves the problem of the admixture being easily spoiled and enables the use-by date of the admixture to be extended.

[0026] <Mixing step of mixing a solid fraction containing spore-forming bacteria with an arbitrary admixture to obtain a bacteria-containing admixture> The solid fraction containing spore-forming bacteria can be mixed with any admixture by a method known to those skilled in the art in accordance with the technical common sense of those skilled in the art to obtain a bacteria-containing admixture. If no admixture is added, the mixing step can be omitted.

[0027] Optional admixtures may include, for example, the following nutrient sources and additives:

[0028] [Nutrition Source] Examples of nutrient sources include organic carbon sources (sugars, starch, etc.), inorganic carbon sources (sodium carbonate, etc.), organic nitrogen sources (amino acids, peptone, etc.), inorganic nitrogen sources (ammonium salts, nitrates, etc.), and inorganic nutrient sources (P, S, K, Mg, Fe, Na, etc.), and one or more of these can be used. However, among the inorganic nutrient sources, inorganic nutrient sources contained in cement, such as calcium, do not need to be added separately.

[0029] The nutrient source is preferably one that does not release corrosive substances through the metabolism of aerobic microorganisms. If the carbon source (sugars, etc.) used as the nutrient source causes corrosive substances such as organic acids (acetic acid, pyruvic acid), a nitrogen source may be added to the nutrient source to mask the organic acids with ammonia produced by the microorganisms.

[0030] Germination inducers can also be added as a nutrient source. The germination inducers are not particularly limited, but examples thereof include amino acids such as L-alanine and L-valine.

[0031] Furthermore, polyhydroxyalkanoic acids (PHAs) produced by biotechnology can be used as a nutrient source. Examples of polyhydroxyalkanoic acids (PHAs) produced by biotechnology include, but are not limited to, polyhydroxybutanoic acid (PHB), poly(3-hydroxybutanoic acid / 3-hydroxyvaleric acid) (PHBV), and poly(3-hydroxybutanoic acid / 3-hydroxyhexanoic acid) (PHBH). Among these, considering that they are rapidly decomposed, easily assimilated, and are soft and easily absorbed by microorganisms, it is preferable that the polyhydroxyalkanoic acid (PHAs) produced by biotechnology are polyhydroxyalkanoic acids (PHAs) selected from the group consisting of poly(3-hydroxybutanoic acid / 3-hydroxyvaleric acid) (PHBV), poly(3-hydroxybutanoic acid / 3-hydroxyhexanoic acid) (PHBH), and mixtures thereof. Commercially available bioengineered PHAs are available from, for example, Kaneka (PHBH) and HighChem (PHBV).

[0032] [Additives] Additives other than nutrient sources can be added to the bacteria-containing admixture of the present invention. The additives are not particularly limited, and various additives can be used, such as reducing agents, fillers, dispersants, surfactants, pH adjusters, and pH buffers, as long as they do not inactivate the activity of aerobic spore-forming bacteria. Only one type of additive can be used alone, or two or more types can be used. The blending ratio of the additives can be appropriately determined based on known usage amounts.

[0033] A culture medium (for example, yeast extract agar (YE) or LB medium) can be added to the bacteria-containing admixture of the present invention as a solution for dissolving the solid fraction (including spore-forming bacteria).

[0034] To the bacteria-containing admixture of the present invention, materials can be appropriately selected and added from the components (for example, admixtures) used in cement compositions described below.

[0035] [Mixing ratio of optional admixtures] The mixing ratio of any admixture can be appropriately determined according to the common technical knowledge of a person skilled in the art, taking into consideration the target mixing ratio of each component in the cement composition.

[0036] [Number of spore-forming bacteria in the admixture] The number of spore-forming bacteria in the admixture can be determined appropriately according to the common technical knowledge of a person skilled in the art, taking into consideration the target number of spore-forming bacteria in the cement composition. 3 10 per 13 If the target number of spore-forming bacteria is 10 per mL, the number of spore-forming bacteria in the admixture should be 10 10 ~10 12 (Preferably, 10 11 ) and the admixture is added to 1 m of cement composition. 3 This target can be achieved by adding 10 mL to 1000 mL (preferably 100 mL) to the solution.

[0037] [Bacteria-containing admixture obtained by the method for producing a bacteria-containing admixture of the present invention] The bacteria-containing admixture obtained by the method for producing a bacteria-containing admixture of the present invention may be in either liquid or solid (dried) form. For example, a liquid form can be obtained by dissolving or dispersing a solid fraction containing spore-forming bacteria, and optional nutrient sources and additives as admixtures in a medium such as water or an organic solvent. The medium preferably contains water, more preferably contains water as the main component (50% by mass or more), and more preferably is composed of water.

[0038] <Method for manufacturing admixture including medium exchange step> In the method for producing a bacteria-containing admixture of the present invention, after the solid-liquid separation step, a medium exchange step can be performed in which the solid fraction containing the spore-forming bacteria is added to a new medium (i.e., the medium in which the aerobic spore-forming bacteria was cultured is exchanged for a new medium) to obtain a mixture of the solid fraction and the new medium. This medium exchange step includes a step of mixing the mixture of the solid fraction and the new medium with an arbitrary admixture to obtain a bacteria-containing admixture.

[0039] [Culture exchange step for obtaining a mixture of a solid fraction containing spore-forming bacteria and a new culture medium] A mixture of the solid fraction and the novel medium can be obtained by mixing the solid fraction and the novel medium using a known mixing method (for example, a mixing method using a stirrer) in accordance with the technical common sense of those skilled in the art.

[0040] The mixing ratio of the solid fraction and the new medium can be appropriately determined according to the common technical knowledge of a person skilled in the art, taking into consideration the target number of spore-forming bacteria in the cement composition.

[0041] Examples of novel media include LB medium and yeast extract medium, with yeast extract medium being preferred.

[0042] [Mixing step of mixing the mixture of the solid fraction containing spore-forming bacteria and the new medium with any admixture to obtain a bacteria-containing admixture] A bacteria-containing admixture can be obtained by mixing a mixture of a solid fraction containing spore-forming bacteria and a new medium with any admixture by a known method according to the common technical knowledge of a person skilled in the art. Note that if no admixture is added, the mixing step can be omitted.

[0043] The types of admixtures, the mixing ratios of the admixtures, etc. are as explained above in <Mixing step of mixing a solid fraction containing spore-forming bacteria with any admixture to obtain a bacteria-containing admixture>.

[0044] The use of a mixture of a solid fraction containing spore-forming bacteria and a novel medium can improve the dispersibility of admixtures in cement compositions. In addition, the addition of spore-forming bacteria to the novel medium can provide a favorable environment for the spore-forming bacteria.

[0045] <Method for manufacturing admixtures including a refrigerated storage process> The method for producing the bacteria-containing admixture of the present invention can include, after the solid-liquid separation step, a refrigerated storage step of storing the solid fraction or the mixture of the solid fraction and the new medium in a refrigerator.

[0046] [Refrigerated storage step of storing the solid fraction or the mixture of the solid fraction and the new medium in a refrigerator] The refrigerated storage conditions can be appropriately determined taking into consideration the conditions under which spore-forming bacteria form spores. For example, the refrigeration temperature is preferably in the range of 4 to 10° C. The refrigeration time is preferably at least 24 hours or more.

[0047] This refrigerated storage process eliminates the risk of many spore-forming bacteria dying due to the mechanical stresses that occur during concrete mixing. Specifically, this refrigerated storage process causes some of the spore-forming bacteria to form spores (covered in a cortex or spore coat that is resistant to external stress), which allows the spore-forming bacteria to withstand the stresses that occur during mixing, allowing more spore-forming bacteria to survive.

[0048] <Method of manufacturing cement composition> A cement composition can be produced using the bacteria-containing admixture obtained by the method for producing a bacteria-containing admixture of the present invention. The method for producing a cement composition includes, for example, a mixing step of mixing at least four materials for a cement composition, namely, the bacteria-containing admixture obtained by the method for producing a bacteria-containing admixture of the present invention, cement, water, and an admixture (or at least five materials for a cement composition, namely, the bacteria-containing admixture obtained by the method for producing a bacteria-containing admixture of the present invention, cement, water, an admixture, and an admixture for a cement composition described below) to obtain a cement composition.

[0049] The mixing step can be carried out by a known method according to the common technical knowledge of a person skilled in the art.

[0050] [cement] Cement is an inorganic binder that hardens when mixed with water, and hydraulic cement is used in the present invention. As hydraulic cement, simple cement such as Portland cement (JIS R5210), hydraulic lime, Roman cement, or natural cement may be used, or mixed cement such as lime-blended cement or mixed Portland cement (JIS R5211, R5212, R5213) may be used.

[0051] [water] The water to be mixed in the cement composition is not limited to pure water, but tap water, river water, lake water, and seawater can also be used.

[0052] [Optional ingredients] The cement composition may also contain aggregate as an optional component. Aggregate is generally used in the production of concrete and is added to suppress heat generation due to the hydration reaction of the cement composition, suppress shrinkage, and reduce the amount of cement used to reduce costs. Aggregate is divided into coarse aggregate and fine aggregate, with 85% or more of the coarse aggregate retained by a 5 mm mesh sieve by mass, and fine aggregate passing through a 5 mm mesh sieve and 100% passing through a 10 mm mesh sieve by mass.

[0053] [Admixture] The cement composition may also optionally contain admixtures such as air entraining agents (air entraining agents), water reducing agents (air entraining water reducing agents, water reducing agents, high performance air entraining water reducing agents, etc.), fluidizing agents, setting / hardening regulators, accelerators, rust inhibitors, waterproofing agents, and antifoaming agents as admixtures for cement compositions.

[0054] [Admixtures for cement compositions] Any admixture (e.g., any admixture not used in the method for producing a bacteria-containing admixture of the present invention) used in the above-mentioned <mixing step of mixing the solid fraction containing spore-forming bacteria with any admixture to obtain a bacteria-containing admixture> can be used in a cement composition as an admixture for a cement composition.

[0055] [Amount of each material in the cement composition] The amount of each material (e.g., spore-forming bacteria, nutrient source for spore-forming bacteria, water, cement, fine aggregate, coarse aggregate) in the cement composition can be appropriately determined in accordance with the common technical knowledge of those skilled in the art and the examples described below.

[0056] When a solid fraction containing spore-forming bacteria is used, the spore-forming bacteria in the cement composition may be, for example, 1 ml of the cement composition. 3 10 per 10 ~10 15 , preferably 10 12 ~10 13 When a mixture of a solid fraction containing spore-forming bacteria and a new medium is used, the mixture is blended in an amount of, for example, 1 to 50% and preferably 5 to 30% relative to the amount of water.

[0057] The nutrient source for spore-forming bacteria in the cement composition is mixed in an amount of, for example, 0.1 to 0.5%, preferably 0.2 to 0.4%, relative to the amount of water. The medium as a solution for dissolving the solid fraction containing spore-forming bacteria in the cement composition is mixed in an amount of, for example, 0.01 to 0.2%, preferably 0.01 to 0.1%, relative to the amount of water.

[0058] The method for producing the cement composition of the present invention includes, for example, powder-mixing cement and any other powder components, adding water and the bacteria-containing admixture obtained by the method for producing the bacteria-containing admixture of the present invention to the powder mixture, kneading the mixture to form a paste, and mixing fine aggregate and coarse aggregate as necessary.

[0059] <Method of manufacturing hardened cement> A hardened cement body can be produced using the bacteria-containing admixture obtained by the method for producing a bacteria-containing admixture of the present invention. Specifically, the method for producing a hardened cement body includes a preparation step of preparing a cement composition by the above-mentioned method for producing a cement composition, and a curing step of curing the cement composition to obtain a hardened cement body.

[0060] [Curing step of curing the cement composition to obtain a hardened cement body] In this curing step, the cement composition is cured to obtain a hardened cement body. The cement composition is applied to a steel material, sprayed, and / or poured into a formwork, and cured until the required compressive strength is obtained, thereby hardening the composition to obtain a hardened cement body. Curing can be carried out under conventionally known conditions, such as air curing or underwater curing.

[0061] The concept of a hardened cement body obtained by the method for producing a hardened cement body includes, for example, cement paste obtained by hardening a cement composition whose main components are cement, water, and admixtures, mortar obtained by hardening a cement composition whose main components are cement, water, fine aggregate, and admixtures, and concrete obtained by hardening a cement composition whose main components are cement, water, fine aggregate, coarse aggregate, and admixtures. [Example]

[0062] EXAMPLES The present invention will be specifically explained below by showing examples, but the present invention is not limited to these examples.

[0063] <Evaluation of the quality of cement compositions and hardened cement bodies when solid fractions containing spore-forming bacteria are used in cement compositions> [Growth of spore-forming bacteria] The AH2 strain was used as a spore-forming bacterium and was grown by the following pre-culture and main culture.

[0064] (preculture) The AH2 strain was used as a spore-forming bacterium. 0.8 mL of the AH2 strain stock solution, 0.5 g of LB medium (Miller), and 20 mL of distilled water were dispensed into a test tube, covered with commercially available food wrap, and cultured overnight at 30°C with shaking at 100 rpm. Note that a 50 mL volume of the AH2 strain stock solution contains 800 to 1 billion AH2 strains.

[0065] The material composition of the LB medium (Miller) was heptone (milk casein) [10 g / L], yeast extract [5 g / L], and sodium chloride [10 g / L].

[0066] (Main culture) 100 ml of the AH2 strain obtained in the preculture was inoculated into 4 L of yeast extract agar (YE) and cultured at 30°C with shaking at 100 rpm for 16 to 24 hours. The bacteria cultured in the preculture were cultured in an amount of yeast extract agar that was 0.8 times the amount of LB medium (Miller) used in the preculture (0.8 x YE) (specifically, 4 g of yeast extract medium was used for 5 g of LB medium (Miller)). The culture solution obtained in the main culture contained 1 to 2 trillion AH2 strain cells.

[0067] [Solid-liquid separation to separate the solid fraction containing the AH2 strain from the liquid fraction containing the culture metabolites] The culture medium obtained by the main culture was separated into a solid fraction containing the AH2 strain remaining on the membrane filter and a liquid fraction containing the culture metabolic solution that passed through the membrane filter using a membrane filter unit manufactured by TPP (product name: Rapid Filter Max Set (bottle top & bottle) 1000 ml; PES membrane filter with a pore size of 0.2 μm). The solid fraction containing the AH2 strain was used in the cement composition.

[0068] [Evaluation of cement composition quality] (Preparation of cement composition) The cement compositions were prepared according to the formulation shown in Table 1 below. Specifically, the mixture consisted of water (Kikugawa City tap water: indicated by "W" in Table 1), cement (high-early-strength Portland cement, density 3.14 g / cm 3 , Taiheiyo Cement Corporation: indicated as "C(H)" in Table 1), fine aggregate (mountain sand, surface dry density 2.61 g / cm 3 , SK Material Co., Ltd.: indicated by "S" in Table 1), coarse aggregate (mountain gravel, surface dry density 2.65 g / cm 3 , SK Materials: indicated by "G" in Table 1), high-performance water reducer (Masterglanium 8000P, Pozzolith Solutions: classified as "AD" in Table 1 (note that "AD" stands for admixture)), air-entraining agent (MasterAir 202, Pozzolith Solutions: classified as "AD" in Table 1), defoamer (MasterAir 404, Pozzolith Solutions: classified as "AD" in Table 1), The solid fraction containing the AH2 strain (which belongs to "AD" in Table 1), yeast extract medium (used as a dissolving solution for the solid fraction containing the AH2 strain; designated "YE" in Table 1; which belongs to "AD" in Table 1), and biodegradable plastic powder (manufactured by HighChem: designated "PHBV" in Table 1; which belongs to "AD" in Table 1) were mixed and kneaded for 2 minutes in a forced twin-shaft mixer to prepare the cement compositions (kneaded products) of Reference Example 1 and Examples 1 to 4.

[0069] (Methods and results for measuring the quality of cement compositions) The quality (fresh properties) of each cement composition was measured, specifically, for slump, air content, and concrete temperature. The slump was measured in accordance with JIS A 1101 for the cement composition (kneaded product) immediately after mixing. The air content was measured in accordance with JIS A 1128 for the cement composition (kneaded product) immediately after mixing. The concrete temperature was measured in accordance with JIS A 1156. The results are shown in Table 1 below.

[0070] [Evaluation of hardened cement paste (specimen)] (Manufacturing of hardened cement (test specimen)) The obtained cement compositions of Reference Example and Examples 1 to 4 were each cast into a cylindrical formwork measuring 10 cm in diameter and 20 cm in height, and compacted using a ram. Each of the cement compositions of Reference Example and Examples 1 to 4 contains fine aggregate and coarse aggregate in addition to water and cement, so the hardened cement products obtained from these cement compositions are specifically concrete. The hardened cement products (concrete) were produced in accordance with JIS A 1132 "Method of producing specimens for concrete strength tests."

[0071] After casting, the concrete was demolded one day later and subjected to standard underwater curing. The curing was carried out in water at 20°C, and test specimens were obtained. Three test specimens were prepared for each cement composition on the 28th day of curing (28-day material age).

[0072] (Measurement method and results of hardened cement paste (specimen)) Three specimens for each cement composition were subjected to a compressive strength test, which was conducted in accordance with JIS A 1108:2018 "Testing method for compressive strength of concrete."

[0073] For each cement composition, a compressive strength test was carried out on three specimens at 28 days old, and the average value was taken as the compressive strength. The results are shown in Table 1 below.

[0074] [Table 1]

[0075] W / C (%) is the mass ratio percentage of unit water (W) to unit cement (C). s / a (%) is the percentage of the volume ratio of fine aggregate volume (s) to aggregate volume (a). In the AE agent, 5A means C x 0.005%, and 7A means C x 0.007%. In antifoaming agents, 4T means C x 0.004%. The specified amount of the solid fraction containing the AH2 strain is the amount of the AH2 strain per 1 ml of cement composition. 3 10 per 13 It means the quantity of an individual.

[0076] The nutrient source of the solid fraction containing the AH2 strain was the usual amount (W × 0.4% and 2 kg / m 3 ), there was almost no effect on the slump properties, and adjustment of the unit water content and the amount of AE water-reducing agent added was not necessary. Furthermore, the effect on the air content was slight, and no antifoaming agent was required, and the specified air content could be achieved simply by adjusting the AE agent, which is commonly done. Furthermore, when the amount of nutrient source added was extremely large (4 kg / m 3 ) required a small amount of antifoaming agent.

[0077] <Evaluation of the quality of cement compositions and hardened cement bodies when a culture medium obtained by replacing a solid fraction containing spore-forming bacteria is used in cement compositions> [Growth of spore-forming bacteria] The AH2 strain was grown in the same manner as in [Growth of spore-forming bacteria] in the above section <Evaluation of the quality of cement compositions and hardened cement products when solid fractions containing spore-forming bacteria are used in cement compositions>.

[0078] [Solid-liquid separation to separate the solid fraction containing the AH2 strain from the liquid fraction containing the culture metabolites] A solid fraction containing the AH2 strain was obtained using the same method as in the "Solid-liquid separation to separate a solid fraction containing the AH2 strain and a liquid fraction containing the culture metabolic solution" in <Evaluation of the quality of cement compositions and hardened cement bodies when solid fractions containing spore-forming bacteria are used in cement compositions> above.

[0079] [Changing the medium to obtain a mixture of the solid fraction containing the AH2 strain and new medium] The solid fraction containing strain AH2 obtained in the above [Solid-liquid separation to separate the solid fraction containing strain AH2 from the liquid fraction containing the culture metabolites] was resuspended in fresh yeast extract medium (YE) so that the volume of fresh yeast extract medium was 0.8 times (0.8 x YE) the volume of LB medium (Miller) used in [Preparation of the solid fraction containing spore-forming bacteria].

[0080] This resuspension yielded 4 liters of a mixture of the solid fraction containing the AH2 strain and the yeast extract medium. The cell count of the AH2 strain was 4 x 10 8 The number was 1 / ml.

[0081] [Evaluation of cement composition quality] (Preparation of cement composition) The cement compositions were prepared according to the formulations shown in Table 2 below. The material names in Table 2 are the same as those in Table 1. In Table 2, "C(BB)" indicates cement (blast furnace cement type B (BB), density 3.04 g / cm 3 , manufactured by Sumitomo Osaka Cement Co., Ltd.), and AE water-reducing agent (Master Pozzolith No. 70, manufactured by Pozzolith Solutions Co., Ltd.: belongs to "AD" in Table 1.

[0082] The materials in Table 2 were mixed and kneaded for 2 minutes in a forced twin-shaft mixer to prepare cement compositions (kneaded products) of Reference Example 2 and Examples 5 to 8.

[0083] (Methods and results for measuring the quality of cement compositions) The quality (fresh properties) of each cement composition was measured, specifically, by measuring the slump, air content, and concrete temperature. The methods for measuring the slump, air content, and concrete temperature were the same as those described above in <Evaluation of the quality of cement compositions and hardened cement products when a solid fraction containing spore-forming bacteria is used in cement compositions>. The results are shown in Table 2.

[0084] [Evaluation of hardened cement paste (specimen)] The evaluation method of the hardened cement product (specimen) was the same as that described above in <Evaluation of the quality of cement compositions and hardened cement products when solid fractions containing spore-forming bacteria are used in cement compositions>. The results are shown in Table 2.

[0085] [Table 2]

[0086] W / C (%) is the mass ratio percentage of unit water (W) to unit cement (C). s / a (%) is the percentage of the volume ratio of fine aggregate volume (s) to aggregate volume (a). In the AE agent, 2A means C x 0.002%, 3A means C x 0.003%, and 4A means C x 0.001%.

[0087] Even when the mixed solution of the solid fraction containing the AH2 strain and new medium was replaced with up to 30% of the unit water volume (W), there was almost no effect on the slump properties, and adjustments to the unit water volume and the amount of AE water-reducing agent added were not necessary. Furthermore, the effect on the air content was negligible, and no antifoaming agent was required; the specified air content could be achieved simply by adjusting the AE agent, as is commonly done.

[0088] <Evaluation of the quality of cement compositions and hardened cement bodies when culture broth of spore-forming bacteria is used in the conventional manner> [Growth of spore-forming bacteria] The AH2 strain was used as a spore-forming bacterium and grown in the same manner as in (pre-culture) and (main culture) in the above <Evaluation of the quality of cement compositions and hardened cement bodies when solid fractions containing spore-forming bacteria are used in cement compositions>.

[0089] [Evaluation of cement composition quality] (Preparation of cement composition) The cement compositions were prepared according to the formulations shown in Table 3 below. The material names in Table 3 are the same as those in Table 2.

[0090] The materials in Table 3 were mixed and kneaded for 2 minutes in a forced twin-axis mixer to prepare cement compositions (kneaded products) of Comparative Examples 1 to 4.

[0091] (Methods and results for measuring the quality of cement compositions) The quality (fresh properties) of each cement composition was measured, specifically, by measuring the slump, air content, and concrete temperature. The methods for measuring the slump, air content, and concrete temperature were the same as those described above in <Evaluation of the quality of cement compositions and hardened cement products when a solid fraction containing spore-forming bacteria is used in cement compositions>. The results are shown in Table 3 below.

[0092] [Evaluation of hardened cement paste (specimen)] The evaluation method of the hardened cement product (specimen) was the same as that described above in <Evaluation of the quality of cement compositions and hardened cement products when solid fractions containing spore-forming bacteria are used in cement compositions>. The results are shown in Table 3 below.

[0093] [Table 3]

[0094] W / C (%) is the mass ratio percentage of unit water (W) to unit cement (C). s / a (%) is the percentage of the volume ratio of fine aggregate volume (s) to aggregate volume (a). In the AE agent, 3A means C x 0.003%, and 4A means C x 0.001%. In antifoaming agents, 3T means C x 0.003%, 5T means C x 0.005%, and 7T means C x 0.007%.

[0095] When a culture medium of spore-forming bacteria was used in a conventional manner, the fresh properties of the cement composition and the compressive strength of the hardened cement body could not be maintained unless an antifoaming agent was added.

Claims

1. A method for producing a bacteria-containing admixture for addition to a cement composition, comprising: a step of culturing and growing aerobic spore-forming bacteria; a solid-liquid separation step of separating the culture solution in which the aerobic spore-forming bacteria have been grown into a solid fraction containing the spore-forming bacteria and a liquid fraction containing a culture metabolic solution; a mixing step of mixing the solid fraction containing the spore-forming bacteria with an arbitrary admixture to obtain a bacteria-containing admixture; A method for producing a bacteria-containing admixture for addition to a cement composition, comprising:

2. After the solid-liquid separation step, the solid fraction containing the spore-forming bacteria is added to a new medium, and a medium exchange step is performed to obtain a mixed solution of the solid fraction and the new medium.

2. The method for producing a bacteria-containing admixture for addition to a cement composition according to claim 1, characterized in that the mixing step is a step of mixing a mixture of the solid fraction containing the spore-forming bacteria and a new medium with an arbitrary admixture to obtain a bacteria-containing admixture.

3. 3. A method for producing a bacteria-containing admixture for addition to a cement composition according to claim 1 or 2, characterized in that after the solid-liquid separation step, a refrigerated storage step is included in which the solid fraction or a mixed liquid of the solid fraction and a new culture medium is refrigerated.

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