Novel microorganism, admixture containing the novel microorganism, cement composition containing the admixture, hardened cement product of the cement composition, and method for producing hardened cement product using the novel microorganism

Abstract

To provide a new microorganism capable of realizing self-healing of concrete in an earlier stage than before, an admixture containing the new microorganism, a cement composition containing the admixture, a cement hardened body of the cement composition, and a method for producing the cement hardened body using the new microorganism.SOLUTION: The new microorganisms have alkali resistance and proliferation potency in pH11 or above and belong to the genus Bacillus. The admixture is to be added to the cementitious composition and comprises the microorganism. The cement composition comprises the admixture, cement and water. The cement hardened body is obtained by hardening the cement composition. The method for producing the cement hardened body using the new microorganism includes a preparation process for preparing the cement composition and a curing process for obtaining the cement hardened body by curing the cement composition.SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] The present invention relates to a novel microorganism, an admixture containing the novel microorganism, a cement composition containing the admixture, a hardened cement product of the cement composition, and a method for producing the hardened cement product using the novel microorganism. [Background technology]

[0002] Concrete (especially reinforced concrete in which steel materials such as reinforcing bars are embedded in concrete) has excellent performance for architectural applications and is therefore widely used as a building structure.

[0003] 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. In the case of reinforced concrete, this neutralization and the penetration of chloride ions destroy the passivation film (formed by calcium hydroxide) around the steel embedded in the concrete, causing corrosion of the steel and resulting in the loss of the concrete's inherent performance.

[0004] To prevent concrete from losing its inherent performance due to cracks on the surface, for example, Patent Document 1 discloses a method for producing so-called self-healing concrete that contains a cement starting material and a bacterial material. In concrete obtained using this self-healing concrete, when cracks occur in the concrete, the bacteria begin metabolic activity, causing the concrete to autonomously repair the cracks.

[0005] In the prior art such as Patent Document 1, bacteria of the genus Bacillus (for example, a microbial species closely related to Bacillus subtillus natto) are mainly used. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Special Publication No. 2013-523590 Summary of the Invention [Problem to be solved by the invention]

[0007] However, the self-healing concrete that uses bacteria in conventional technologies such as Patent Document 1 has the disadvantage that the concrete will not autonomously repair cracks unless cracks appear on the concrete surface due to deterioration over time, or unless the cracks cause neutralization.

[0008] It is desirable to use bacteria within the concrete to self-heal the concrete before deterioration over time progresses seriously, for example, before the concrete surface is severely damaged by an earthquake, or before the oxygen trapped within the concrete during manufacturing severely rusts the steel. However, since Bacillus bacteria do not normally carry out cellular activity unless the concrete's pH shifts from alkaline to acidic due to cracks or other factors, self-healing of the concrete does not occur unless the phenomenon of neutralization occurs.

[0009] As described above, even with the self-healing concrete using bacteria in the prior art such as Patent Document 1, there still remain problems to be solved.

[0010] SUMMARY OF THE INVENTION In view of the above problems, an object of the present invention is to provide a self-healing concrete that can achieve self-healing at an earlier stage than conventional concrete. [Means for solving the problem]

[0011] The present inventors have conducted extensive research to achieve the above object, and as a result have found that the above object can be achieved by using a microorganism belonging to the genus Bacillus, which has alkali resistance and the ability to grow under highly alkaline conditions, in a concrete material, thereby completing the present invention.

[0012] The microorganism of the invention described in claim 1 for achieving the above object is characterized in that it has alkali resistance and the ability to grow at a pH of 11 or higher, and belongs to the genus Bacillus.

[0013] The invention described in claim 2 is characterized in that, in the invention described in claim 1, the bacterium belongs to Bacillus altitudinis.

[0014] The invention described in claim 3 is characterized in that, in the invention described in claim 1, the microorganism is the Bacillus altitudinis AH1 strain deposited at the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation under accession number NITE P-03987.

[0015] The invention described in claim 4 is characterized in that, in the invention described in claim 1, the microorganism is the Bacillus altitudinis AH2 strain deposited at the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation under accession number NITE P-03988.

[0016] The admixture for addition to a cement composition according to the invention described in claim 5 is characterized by containing the microorganism described in claim 1.

[0017] A cement composition according to a sixth aspect of the present invention is characterized by comprising the admixture according to the fifth aspect, cement, and water.

[0018] A hardened cement product of the invention described in claim 7 is characterized by being obtained by hardening the cement composition described in claim 6.

[0019] The method for producing a hardened cement product according to the invention of claim 8 comprises the steps of: A preparation step of preparing the cement composition according to claim 6; a curing step of curing the cement composition to obtain a hardened cement body; The present invention is characterized by comprising: [Effects of the Invention]

[0020] According to the novel microorganism of the present invention, an admixture containing the novel microorganism, a cement composition containing the admixture, a hardened cement product of the cement composition, and a method for producing a hardened cement product using the novel microorganism, it is possible to provide self-healing concrete that can achieve self-healing at an earlier stage than conventional methods. [Brief explanation of the drawings]

[0021] [Figure 1] This figure shows the results of comparing the assembled sequences of the AH1 strain and the AH2 strain using Mummer [version 3.23] (options: -mum -b -c). [Figure 2] This figure shows the results of comparing the gene positions between the assembled sequences of the AH1 strain and the AH2 strain, using Pygernomeviz [version 0.4.4]. [Figure 3] FIG. 1 shows the results of a culture experiment to confirm the pH tolerance of Bacillus subtilis natto. [Figure 4] FIG. 1 shows the results of a culture experiment to confirm the pH tolerance of the AH1 strain. [Figure 5] FIG. 1 shows the results of a culture experiment to confirm the pH tolerance of the AH2 strain. [Figure 6] FIG. 1 shows the results of fluorescence microscopy observations when the number of AH2 strains in concrete was 20% and 30%. [Figure 7] FIG. 1 shows the results of culturing bleeding water on a yeast extract agar medium adjusted to pH 11. DETAILED DESCRIPTION OF THE INVENTION

[0022] <Microorganisms> The microorganism of the present invention has alkali resistance and the ability to grow at a pH of 11 or higher, and belongs to the genus Bacillus.

[0023] [Types of microorganisms] The microorganism of the present invention belongs to the genus Bacillus (which is an aerobic microorganism), and is preferably Bacillus altitudinis.

[0024] The microorganism of the present invention is particularly the Bacillus altitudinis AH1 strain deposited at the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation under accession number NITE P-03987.

[0025] The nucleotide sequence of the 16S rRNA gene of the Bacillus altitudinis AH1 strain (SEQ ID NO: 1) was determined using the analytical equipment used in the <Genome analysis of the microorganism of the present invention> in the Examples described below, and was found to be as shown in Table 1. This nucleotide sequence is the same as the nucleotide sequence in the "Sequence Listing" column described below.

[0026] [Table 1]

[0027] The microorganism of the present invention is the Bacillus altitudinis AH2 strain deposited at the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation under accession number NITE P-03988.

[0028] The nucleotide sequence of the 16S rRNA gene of the Bacillus altitudinis AH2 strain (SEQ ID NO: 2) was analyzed by "<Genome analysis of the microorganism of the present invention>" in the Examples described below, and the result is shown in the following Table 2. This nucleotide sequence is the same as the nucleotide sequence in the "Sequence Listing" column described below.

[0029] [Table 2]

[0030] [Genome analysis of the microorganism of the present invention] Genome analysis of the microorganism of the present invention revealed that it belongs to Bacillus altitudinis. Details of this genome analysis are explained in the section "<Genome analysis of the microorganism of the present invention>" in the Examples below.

[0031] [pH value] The microorganism of the present invention has alkali resistance and proliferation ability even at a pH of 11 or higher. The pH value can be a range with only a lower limit selected from 11, 11.5, 12, 12.5, 13, 13.5, or 14, or a range with both a lower limit and an upper limit selected from the above values. For example, a lower limit of 12 can be selected to achieve a pH of 12 or higher. Alternatively, a lower limit of 11 and an upper limit of 13 can be selected to achieve a range of 11 to 13.

[0032] [Function of the microorganisms of the present invention on concrete] As shown in the section "Confirmation of pH Tolerance of Microorganisms" in the Examples below, the microorganisms of the present invention have alkali resistance and proliferation ability even under highly alkaline conditions of pH 11 or higher, and can function under highly alkaline conditions. In other words, the microorganisms of the present invention can actively utilize the water and oxygen in the concrete to promote the mineralization reaction, even in the absence of the phenomenon of neutralization in which the pH of the concrete shifts from alkaline to acidic (for example, due to cracks in the concrete). Specifically, the microorganisms of the present invention can digest oxygen and emit carbon dioxide even under highly alkaline conditions, thereby promoting the mineralization reaction and accelerating (strengthening) the reaction that hardens the concrete itself.

[0033] Furthermore, since the microorganisms of the present invention can be active even under highly alkaline conditions, they can cause the concrete to exhibit self-healing properties at an early stage before the phenomenon of neutralization, in which the pH of the concrete shifts from alkaline to acidic, occurs.

[0034] In addition, the microorganisms of the present invention have the ability to consume and remove oxygen, which causes rust, even under highly alkaline conditions, and therefore can exert their rust-preventing function before the oxygen trapped in the concrete during concrete production can seriously rust the steel material.

[0035] <Mixed materials> The admixture of the present invention is added to a cement composition and contains the microorganism of the present invention. The admixture of the present invention may also contain a nutrient source.

[0036] [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.

[0037] 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.

[0038] 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.

[0039] The nutrient sources mentioned above include those that affect the strength development of the hardened cement body, and therefore the mixing ratio thereof is, for example, 10% or less, preferably 5% or less by mass, based on the total mass of the admixture excluding water.

[0040] 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).

[0041] [Additives] Additives other than nutrient sources can be added to the 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 microorganisms. 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 amounts.

[0042] [Mold of admixture] The admixture of the present invention may be in either liquid or solid (dry) form. In the case of a liquid form, the microorganism of the present invention and optional nutrient sources and additives are dissolved or dispersed in a medium such as water or an organic solvent to form a liquid. 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.

[0043] <Cement composition> The cement composition of the present invention contains the admixture of the present invention, cement, and water. The admixture is the same as the admixture described above, so a description thereof will be omitted here.

[0044] The amount of the admixture of the present invention in the cement composition is, for example, 3 The number of microbial cells per 10 ~10 15 / m 3 The amount of the mixture is 10 12 ~10 13 / m 3 It is preferable that the blending amount is such that:

[0045] The amount of the admixture of the present invention in the cement composition is, for example, 3 The amount of biotechnologically produced polyhydroxyalkanoates (PHA) per unit volume is 0.3-10 kg / m 3 The blending amount is 0.5 to 8 kg / m 3 It is preferable that the blending amount is such that:

[0046] [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.

[0047] [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.

[0048] [Optional ingredients] The cement composition also contains 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. Coarse aggregate is one that retains 85% or more by mass on a 5 mm mesh sieve, while fine aggregate is one that passes through a 5 mm mesh sieve and 100% by mass on a 10 mm mesh sieve.

[0049] It may also contain optional 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.), superplasticizers, setting and hardening regulators, accelerators, rust inhibitors, waterproofing agents, etc.

[0050] The cement composition can be prepared, for example, by powder-mixing cement and, optionally, other powder components, adding water and the admixture of the present invention to the powder mixture, kneading the mixture to form a paste, and then mixing fine aggregate and coarse aggregate as necessary.

[0051] <Hardened cement> The hardened cement product of the present invention can be obtained by hardening the cement composition of the present invention by a known method.

[0052] Specifically, the prepared cement composition is poured into a suitably prepared formwork and cured. The formwork preferably contains reinforcing bars, but may not contain reinforcing bars. The cement composition poured into the formwork is then compacted to remove air bubbles. Compaction is carried out by vibration compaction using a vibrator. After compaction, the composition is cured until the required compressive strength is obtained, and a hardened cement body is obtained.

[0053] The concept of the hardened cement product of the present invention includes, for example, concepts of 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.

[0054] <Method of manufacturing hardened cement> The method for producing a hardened cement product of the present invention includes a preparation step of preparing the cement composition of the present invention, and a curing step of curing the cement composition to obtain a hardened cement product.

[0055] [Preparation step of preparing the cement composition of the present invention] The cement composition in this preparation step can be prepared by a known method based on the common technical knowledge of a person skilled in the art, based on the above descriptions of the <admixture> and <cement composition>.

[0056] The timing for adding the microorganisms may be, for example, the following timing (A) or (B).

[0057] (A): Microorganisms are added when mixing or kneading ingredients other than microorganisms (pre-addition). Examples of this method of addition include adding microorganisms to water, which is an ingredient other than microorganisms, adding microorganisms to cement, which is an ingredient other than microorganisms, or adding microorganisms to a mixture of ingredients other than microorganisms.

[0058] (B): Concrete is prepared in advance by mixing or kneading ingredients other than microorganisms, and then microorganisms are added to the concrete (post-addition). For example, one method of adding microorganisms is to purchase concrete that does not contain microorganisms, transport the concrete to the construction site using an agitator truck, add the microorganisms to the truck, and mix it by turning the drum.

[0059] [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. [Example]

[0060] EXAMPLES The present invention will be specifically explained below by showing examples, but the present invention is not limited to these examples.

[0061] <Genome analysis of the microorganism of the present invention> [Genome analysis method] Genome analysis was performed separately for the AH1 and AH2 strains. Specifically, DNA was extracted using a Quick-DNA MiniPrep Kit (ZYMO RESEARCH). Next, a library was prepared from the extracted genomic DNA using a QIAGEN QIAseq FX DNA Library Kit. After this library preparation, short-read sequencing was performed using an Illumina NextSeq2000. Furthermore, after this library preparation, long-read sequencing was performed using a MinlON (Oxford Nanopore Technologies).

[0062] Next, using free software genome analysis tools such as MaSuRCA, POLCA, and GTDBTk, genome data were assembled and annotated according to the following steps (1) to (10).

[0063] (1) Assembling was performed from short and long reads using MaSuRCA [version 4.1.0]. The specified option was FLYE_ASSEMBLY=1.

[0064] (2) Using Medata [version 1.9.1], the long reads were mapped to the assembled sequence obtained in (1) above, and the assembly was proofread. The specified option was --modelr1041_e82_400bps_sup_v4.2.0. This procedure was repeated three times.

[0065] (3) Using bbduk.sh [version 38.90], we trimmed short read adapter sequences and removed low-quality reads. The specified options were ktrim=r ref=adapters k=23 mink=ll hdist=l tpe tbo qtrim=r rimq=10 minlength=40 maxns=l minavgquality=15.

[0066] (4) Using bbmap.sh [version 38.90], the read sequences were mapped to the masked human genome and the mapped reads were removed as human contaminant reads. The specified options were quickmatch, fast, untrim, minid=0.95, maxindeI=3, bwr=0.16, bw=12, minhits=2, path=human_masked_index. (*) qtrim=rl trimq=10.

[0067] (5) Using Polypolish [version 0.5.0], the QC short reads obtained in (4) above were mapped to the assembled sequence obtained in (2) above and proofread. The options were default.

[0068] (6) Using POLCA [version 4.1.0], the QC short reads obtained in (4) above were mapped to the assembled sequence obtained in (5) above and proofread. The options were default.

[0069] (7) Using Prokka [version 1.14.5], gene regions were estimated from the assembled sequence obtained in (6) above. The specified options were --rawproduct --mincontiglen 1000.

[0070] (8) Using QUAST [v5.0.2], the number of contigs, total length, GC content, etc. of the assembled sequence obtained in (6) above were evaluated. The specified options were the default.

[0071] (9) The completeness and contamination of the assembled sequence obtained in (6) above was evaluated using CheckM [V1.1.3]. The specified options were lineage_wf -r --ali --genes tab_table.

[0072] (10) Using GTDBTk [version 2 3.2], we inferred the phylogenetic information of the assembled sequences obtained in (6) above. The options were default. The database version used for phylogenetic inference was Release 214.

[0073] [Results of short-read sequence analysis, long-read sequence analysis, and hybrid assembly analysis] An overview of the genome quality of the AH1 and AH2 strains based on the results of the above short-read sequence analysis, long-read sequence analysis, and hybrid assembly analysis is shown in Table 3 below.

[0074] As is clear from the "Full taxonomy" column in Table 3, the AH1 and AH2 strains were found to be species closely related to Bacillus altitudinis.

[0075] [Table 3]

[0076] [Comparative genome analysis 1: Sequence comparison (dot plot)] The assembled sequences of strain AH1 and strain AH2 were compared using Mummer [version 3.23] (options: -mum -b -c). The results are shown in Figure 1. As shown in Figure 1, no major insertions, substitutions, or deletions were observed between the two strains.

[0077] [Comparative Genome Analysis 2: Gene Arrangement (Synteny) Analysis] Using Pygernomeviz [version 0.4.4], we compared the gene positions of the AH1 and AH2 strain assembly sequences. The results of this comparison were visualized using the pgv-mmseqs -tick style axis as shown in Figure 2. As is clear from the results shown in Figure 2, the AH1 assembly consists of two contigs. Some of the genes with different sequences were transposon-related.

[0078] [Comparative genome analysis 3: Genes that differ between AH1 and AH2 strains] As a result of gene estimation for the genomic DNA obtained from the AH1 and AH2 strains, the genes that were confirmed to be different between the AH1 and AH2 strains are shown in Table 4 below.

[0079] [Table 4]

[0080] <Confirmation of pH tolerance of microorganisms> In this experiment, a culture experiment was carried out to confirm the pH tolerance of Bacillus subtilis natto, strain AH1, and strain AH2.

[0081] [Preculture] (Natto bacteria) 0.8 mL of stock solution of Bacillus natto (product name: pure culture Bacillus natto, manufactured by Miyagino Natto Seizosho), 0.5 g of LB medium (Miller), and 20 mL of distilled water were dispensed into a test tube, the surface was covered with commercially available food wrap film, and shaking culture was carried out overnight at 30°C and 100 rpm. Note that 50 mL of the stock solution of Bacillus natto contains 300 to 400 million Bacillus natto.

[0082] (AH1 and AH2 strains) 0.8 mL of the AH1 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 with shaking at 100 rpm at 30°C. Note that 50 mL of the AH1 strain stock solution contains 800 to 1 billion AH1 strains.

[0083] The AH2 strain was also pre-cultured in the same manner as the AH1 strain. Note that 50 mL of the AH2 strain stock solution contains 800 to 1 billion AH2 strain cells.

[0084] The material composition of the above-mentioned LB medium (Miller) was heptone (milk casein) [10 g / l], yeast extract [5 g / l], and sodium chloride [10 g / l].

[0085] [Main culture] The Bacillus subtilis natto, AH1 strain, and AH2 strain obtained in the preculture were separately inoculated into yeast extract agar (YE) and cultured overnight at 30°C with shaking at 100 rpm. The cultured bacteria obtained in the preculture were cultured in an amount of yeast extract medium that was 0.8 times the amount of the above-mentioned LB medium (Miller) (0.8 x YE) (specifically, 4 g of yeast extract medium was used for 5 g of LB medium (Miller)). The culture pH was as shown in the "Confirmation of pH tolerance of microorganisms" section below, and the pH was adjusted with sodium hydroxide (1N).

[0086] [Confirmation of pH tolerance of microorganisms] The pH tolerance of each microorganism was confirmed by maintaining a constant culture pH (specifically, the pH was selected from among 9, 10, 11, 11.5, 12, 12.5, and 13) during the main culture and measuring the turbidity versus the culture time.

[0087] The degree of suspension was measured by measuring the absorbance at a wavelength of 660 nm (OD660) using a simple OD monitor (miniphoto 518R) manufactured by Taitec Corporation.

[0088] The results of the culture experiments to confirm the pH tolerance of Bacillus subtilis natto, strain AH1, and strain AH2 are shown in Figures 3 to 5. The graphs of 0.8 x YE in Figures 3 to 5 show the results of the culture experiments when sodium hydroxide for pH adjustment was not added.

[0089] As shown in Figure 3, it was confirmed that Bacillus subtilis var. natto can survive up to pH 10, but does not grow at all above pH 11.

[0090] On the other hand, in the case of the AH1 strain, as shown in FIG. 4, it was confirmed that the optimum pH range was 9 to 10, but that it could grow even at a pH exceeding 11.

[0091] In the case of the AH2 strain, as shown in FIG. 5, the optimum pH range is 9 to 10, but it was confirmed that it can grow even at a pH above 11.

[0092] <Confirmation of the dispersion of microorganisms in concrete> To confirm the dispersibility of microorganisms in concrete, (1) direct counting of microorganisms in bleeding water (squeezed water) by fluorescent observation, and (2) plate culture of bleeding water were carried out.

[0093] [Direct counting of microorganisms in bleeding water by fluorescence observation] After concrete was produced using the AH2 strain, the bleeding water was collected and subjected to fluorescence microscopy observation (total bacterial count measurement) of the AH2 strain. The nucleic acid staining reagent SYBR green I (Life Technologies Corporation) was used for this fluorescence microscopy observation. An all-in-one fluorescence microscope manufactured by Keyence Corporation was used as the fluorescence microscope.

[0094] The concrete was produced by adjusting the ratio of the microbial culture solution to the concrete preparation water to be 5%, 10%, 20%, and 30%.

[0095] Specifically, the concrete was produced by the same methods as those used in the "Preparation of Cement Composition" and "Production of Hardened Cement (Test Specimen)" sections described later in "Confirmation of the Properties of Concrete Containing Microorganisms." The concretes containing 5%, 10%, 20%, and 30% microbial culture solution relative to the concrete preparation water correspond to the concretes obtained using the cement compositions of Examples 1, 2, 3, and 4, described later, respectively. The bleeding water was collected from the water seeping out during the slump measurement for these concretes described later in "Confirmation of the Properties of Concrete Containing Microorganisms."

[0096] The relationship between the number of bacteria administered at each rate and the number of bacteria observed in the breeding water is shown in Table 5 below.

[0097] [Table 5]

[0098] As is clear from the results in Table 5 above, it was confirmed that the number of bacteria administered was about 1 / 10 of the theoretical blend value. In other words, it was found that the AH2 strain was sufficiently dispersed and present in the concrete. It is inferred that the microorganisms will function even if they are mixed into the concrete.

[0099] Fluorescence microscopy observations were performed by adding the nucleic acid staining reagent SYBR green I to the squeezed water, which is believed to contain microorganisms, and staining it, then filtering it and observing the filter under a fluorescence microscope. Figure 6 shows the results of fluorescence microscopy observations when the microbial culture solution in the concrete preparation water was 20% and 30%. As shown in Figure 6, the presence of the AH2 strain in the concrete was confirmed.

[0100] [Plate culture of bleeding water] The bleeding water obtained in the above-mentioned "Direct counting of microorganisms in bleeding water by fluorescent observation" was seeded on an agar medium (yeast extract agar medium (YE)) adjusted to pH 11 using sodium hydroxide (1N), and cultured at a culture temperature of 30°C. The culture results are shown in Figure 7. As shown in Figure 7, germination of the AH2 strain was confirmed, confirming the presence of the AH2 strain in the concrete.

[0101] <Confirming the properties of concrete containing microorganisms> The properties of concrete when the AH2 strain was used as the microorganism were confirmed as follows.

[0102] [Preparation of cement composition] Water (Tsukuba City tap water: indicated as "W" in Table 6), cement (blast furnace cement type B (BB), density 3.04 g / cm 3 , Sumitomo Osaka Cement Co., Ltd.: indicated as "C" in Table 6), fine aggregate (mountain sand, surface dry density 2.58 g / cm 3 , Kakegawa City, Shizuoka Prefecture: indicated by "S" in Table 6), coarse aggregate (crushed stone, surface dry density 2.65 g / cm3 , produced in Sakuragawa City, Ibaraki Prefecture: indicated by "G" in Table 6), an air-entraining water-reducing agent (Master Pozzolith No. 70, manufactured by Pozzolith Solutions: classified as "AD" in Table 6 (note that "AD" stands for admixture)), an air-entraining agent (MasterAir 101, manufactured by Pozzolith Solutions: classified as "AD" in Table 6), an antifoaming agent (MasterAir 404, manufactured by Pozzolith Solutions: classified as "AD" in Table 6), and an AH2 strain microbial culture solution (a microorganism of the present invention: classified as "AD" in Table 6) 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.

[0103] The AH2 strain was cultured using the following method. The cultured bacteria obtained in the "Preculture" section of the "Confirmation of pH Tolerance of Microorganisms" section above were cultured in 0.8xYE liquid medium. The culture was performed by shaking at 30°C and 100 rpm for 16 to 24 hours. The culture was harvested using a membrane filter unit (Rapid Filter Max Set (Bottle Top & Bottle) 1000 ml, manufactured by TPP) and resuspended in fresh 0.8xYE liquid medium to prepare a microbial culture solution. The microbial culture solution contained 1 to 2 trillion AH2 strain cells.

[0104] The blend proportions and qualities (slump, air content, concrete temperature) of each cement composition are shown in Table 6. 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.

[0105] [Table 6]

[0106] [Manufacturing 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 cement and water, and 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."

[0107] 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).

[0108] [Evaluation 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."

[0109] For each cement composition, three specimens were subjected to a compressive strength test at 28 days old, and the average value was recorded as the compressive strength. The results are shown in Table 6 above.

[0110] Comparing the Reference Example with Examples 1 to 3 in Table 6, the concrete not containing the AH2 strain (Reference Example) and the concrete containing the AH2 strain microbial culture solution (Examples 1 to 3) had similar fresh properties and compressive strength. In other words, it was found that even when the AH2 strain microbial culture solution was added to the mixing water, concrete having the same fresh properties and compressive strength as conventional concrete was obtained.

Claims

1. A microorganism characterized by having alkali resistance and proliferation ability at pH 11 or higher and belonging to the genus Bacillus.

2. 2. The microorganism according to claim 1, characterized in that it belongs to the genus Bacillus altitudinis.

3. The microorganism according to claim 1, characterized in that the microorganism is Bacillus altitudinis AH1 strain deposited at the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation under the accession number NITE P-03987.

4. The microorganism according to claim 1, characterized in that the microorganism is Bacillus altitudinis AH2 strain deposited at the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation under the accession number NITE P-03988.

5. An admixture for addition to a cement composition, characterized in that it comprises the microorganism of claim 1.

6. A cement composition comprising the admixture of claim 5, cement, and water.

7. A hardened cement product obtained by hardening the cement composition according to claim 6.

8. A preparation step of preparing the cement composition according to claim 6; a curing step of curing the cement composition to obtain a hardened cement body; A method for producing a hardened cement product, comprising:

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