Method for measuring microbial activity in cement-containing compacts

Abstract

To provide a method for measuring the activity of microorganisms in a cement-containing molded body by which a measurement result can be immediately obtained and analysis can be conducted in an environment where microorganisms are generally present.SOLUTION: A method for measuring the activity of microorganisms in a cement-containing molded body is to measure the degree of activity of microorganisms in a cement-containing molded body obtained by molding a concrete composition including a cement-containing composition, microorganisms, and nutrient sources, wherein at least some of the nutrient sources are isotopically labeled nutrient sources, and the method includes the steps of: releasing, from a surface of the cement-containing molded body, a metabolite generated when the microorganisms included in the cement-containing molded body decompose the isotopically labeled nutrient sources, thereby recovering the metabolite; and measuring an amount of isotopes included in the metabolite, thereby estimating an amount of the isotopically labeled nutrient sources decomposed by the microorganisms.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for measuring microbial activity in a cement-containing molded body. [Background technology]

[0002] By incorporating microorganisms into concrete along with nutrients, it is possible to achieve useful effects such as repairing cracks in the concrete. Research into such crack repair in concrete is being actively conducted. For example, by incorporating bacteria and polylactic acid into concrete materials, self-healing concrete is known, in which when the concrete cracks, the activated bacteria's metabolism precipitates calcium carbonate, repairing the cracks. However, because microbial reactions vary greatly depending on temperature, moisture, and nutrient sources, there are challenges in determining how much of the desired reaction is actually occurring and how much effect the reaction will have.

[0003] One known method for estimating the amount of microbial activity in the environment is to measure the amount of mRNA using quantitative PCR. This method is used to estimate microbial activity in soil during bioremediation and to confirm the activity of microorganisms that decompose target substances. Furthermore, measuring ATP activity is known as a method for rapidly estimating the amount of microbial activity in the environment. Among the methods for measuring ATP activity, for example, the ATP swab test is useful as a means for rapidly measuring the amount of microbial activity on the surface of a test object. In addition, a method for quantitatively determining intracellular metabolic fluxes by conducting tracing experiments with isotope compounds is known, in which the quantitative ratio (carbon balance) of each metabolic product in the metabolic reaction pathway of a target cell is stoichiometrically analyzed (see, for example, Patent Document 1). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 4742528 Summary of the Invention [Problem to be solved by the invention]

[0005] The method of estimating the activity of microorganisms in the environment using quantitative PCR requires many steps for analysis, such as extracting mRNA from soil, treating it with deoxyribonuclease (DNase), and reverse transcription PCR, making it difficult to obtain measurement results immediately. ATP swabbing tests are typically used to test the cleanliness of surfaces after cleaning, and are not suitable for analyzing environments where microorganisms are commonly present, such as concrete surfaces or soil. The method described in Patent Document 1 determines intracellular metabolic flux from analytical values ​​of cells cultured in a medium containing an isotope-labeled substrate as a carbon source, making it difficult to obtain measurement results immediately.

[0006] The present invention has been made in consideration of the above circumstances, and aims to provide a method for measuring microbial activity in a cement-containing molded body, which is capable of obtaining measurement results immediately and is capable of performing analysis in an environment where microorganisms are commonly present. [Means for solving the problem]

[0007] The present invention has the following aspects. [1] A method for measuring the activity of microorganisms in a cement-containing molded body obtained by molding a concrete composition containing a cement-containing composition, microorganisms, and a nutrient source, comprising: At least a portion of the nutrient source is an isotope-labeled nutrient source; a step of releasing metabolic products generated when the microorganisms contained in the cement-containing molded body decompose the isotope-labeled nutrient source from the surface of the cement-containing molded body, and recovering the metabolic products; and estimating the amount of the isotope-labeled nutrient source decomposed by the microorganism by measuring the amount of isotope contained in the metabolic product. [2] The isotope-labeled nutrient source is 13 C-labeled nutrient sources, 15 N-labeled nutrients and 17 The method for measuring microbial activity in a cement-containing molded body according to [1], wherein the microbial activity is at least one selected from the group consisting of O-labeled nutrient sources. [3] The method for measuring microbial activity in a cement-containing molded body according to [1] or [2], wherein the amount of isotopes contained in the metabolites is measured by mass spectrometry of the metabolites. [Effects of the Invention]

[0008] According to the present invention, it is possible to provide a method for measuring microbial activity in a cement-containing molded body, which is capable of obtaining measurement results immediately and is capable of performing analysis in an environment where microorganisms are commonly present. DETAILED DESCRIPTION OF THE INVENTION

[0009] Hereinafter, a method for measuring microbial activity in a cement-containing molded body according to an embodiment of the present invention will be described. It should be noted that the present embodiment is specifically described to allow a better understanding of the gist of the invention, and does not limit the present invention unless otherwise specified.

[0010] [Method for measuring microbial activity in cement-containing compacts] The method for measuring microbial activity in a cement-containing molded body of this embodiment is a method for measuring the activity of microorganisms in a cement-containing molded body obtained by molding a concrete composition containing a cement-containing composition, a microorganism, and a nutrient source, at least a portion of which is an isotope-labeled nutrient source, and includes the steps of: releasing, from the surface of the cement-containing molded body, metabolic products generated when the microorganisms contained in the cement-containing molded body decompose the isotope-labeled nutrient source, and recovering the metabolic products (hereinafter referred to as the "first step"); and measuring the amount of isotope contained in the metabolic products to estimate the amount of the isotope-labeled nutrient source decomposed by the microorganisms (hereinafter referred to as the "second step").

[0011] "Cement-containing compact" The cement-containing molded article in this embodiment is obtained by molding a concrete composition containing a cement-containing composition, microorganisms, and nutrient sources.

[0012] (Cement-containing composition) The cement-containing composition contains cement and water. Examples of cement-containing compositions include concrete, mortar, and cement milk. Concrete is a mixture of cement, fine aggregate (sand), and coarse aggregate (gravel (crushed stone)), mixed with water. Mortar is a mixture of cement and fine aggregate (sand) mixed with water. Cement milk is cement mixed with only water.

[0013] Cement is a powder made primarily from limestone, clay, silica, iron oxide, etc., that hardens through a chemical reaction with water. Fine aggregate is sand with a diameter of 5 mm or less. Coarse aggregate is gravel (crushed stone) with a diameter of more than 5 mm. The diameter of coarse aggregate is preferably 25 mm or less.

[0014] The mixing ratio of cement, fine aggregate, and coarse aggregate in concrete can be determined appropriately depending on the strength required of the concrete. The mixing ratio of cement, fine aggregate, and coarse aggregate, in mass ratio, is preferably, for example, 1 part cement, 2 to 3 parts fine aggregate, and 4 to 6 parts coarse aggregate.

[0015] The mixing ratio of cement to fine aggregate in the mortar can be determined appropriately depending on the strength required for the mortar. The mixing ratio of cement to fine aggregate is preferably, for example, 1 part cement to 2 to 4 parts fine aggregate by mass.

[0016] The water content in the concrete is preferably 6% by mass or more and 15% by mass or less based on the total amount of the concrete. The water content in the mortar is preferably 10% by mass or more and 15% by mass or less based on the total amount of the mortar. The content of water in the cement milk is preferably 30% by mass or more and 40% by mass or less with respect to the total amount of the cement milk.

[0017] The content of the cement-containing composition in the concrete composition is preferably 10% by mass or more and 60% by mass or less based on the total amount of the concrete composition.

[0018] (microorganisms) Examples of microorganisms include bacteria, fungi, and archaea.

[0019] The content of microorganisms in the concrete composition is preferably 0.001% by mass or more and 10% by mass or less, and more preferably 0.005% by mass or more and 5% by mass or less, based on the total amount of the concrete composition. When the content of microorganisms is equal to or more than the lower limit, useful effects for concrete, such as repairing cracks, can be sufficiently achieved. When the content of microorganisms is equal to or less than the upper limit, concrete can be used without impairing its strength.

[0020] (Nutrition source) Examples of nutrient sources include polylactic acid, urea, ethanol, acetic acid, glucose, polypeptide, yeast extract, and the like. At least a portion of the nutrient source is an isotope-labeled nutrient source. For example, when polylactic acid is used as the nutrient source, at least a portion of the polylactic acid is 13 C-labeled polylactic acid.

[0021] The content of the nutrient source in the concrete composition is preferably 0.001% by mass or more and 10% by mass or less, and more preferably 0.005% by mass or more and 5% by mass or less, based on the total amount of the concrete composition. If the content of the nutrient source is equal to or more than the lower limit, useful effects for concrete, such as repairing cracks, can be sufficiently achieved. If the content of the nutrient source is equal to or less than the upper limit, it can be used without impairing the strength of the concrete.

[0022] The mixing ratio of microorganisms and nutrient sources in a concrete composition is, for example, preferably 1 part microorganism to 0.5 to 50 parts nutrient sources by mass, and more preferably 1 to 30 parts nutrient sources. If the ratio of nutrient sources is equal to or greater than the above-mentioned lower limit, the microorganisms can grow with sufficient nutrients, and useful effects for concrete, such as repairing cracks, can be sufficiently achieved. If the ratio of nutrient sources is equal to or less than the above-mentioned upper limit, the amount of nutrient sources remaining in the concrete composition after metabolism by the microorganisms can be reduced, and excessive reactions can be suppressed.

[0023] (Isotope-labeled nutrient sources) The isotope-labeled nutrient source is a compound containing an isotope, which serves as a nutrient source for the microorganism. 12 C) isotope of carbon-13 ( 13 C) labeled nutrients, nitrogen-14( 14 N) isotope of nitrogen-15( 15 N)-labeled nutrients, oxygen-16( 16 Oxygen-17 ( 17 O) Labeled nutrient sources, etc. 13 Examples of C-labeled nutrient sources include: 13 C-labeled polylactic acid, 13 C-labeled urea, 13 C-labeled ethanol, 13 C-labeled acetic acid, 13 Examples include C-labeled glucose. 15 Examples of N-labeled nutrient sources include: 15 N-labeled urea, 15 N-labeled polypeptides and the like. 17 Examples of O-labeled nutrient sources include: 17 O-labeled polylactic acid, 17 O-labeled ethanol, 17 Examples include O-labeled acetic acid. The isotope-labeled nutrient source may be used alone or in combination of two or more kinds.

[0024] 13 Since C-labeled glucose is commercially available, various enzymes were synthesized using this glucose as a carbon source. 13 C-labeled compounds can be prepared. 13 Examples of C-labeled compounds include: 13 C-labeled polylactic acid, 13 C-labeled urea, 13 C-labeled ethanol, 13 Examples include C-labeled acetic acid.

[0025] The content of the isotope-labeled nutrient source is preferably 0.01% by mass or more and 10% by mass or less, and more preferably 0.1% by mass or more and 1% by mass or less, based on the total amount of the nutrient source. If the content of the isotope-labeled nutrient source is equal to or greater than the above-mentioned lower limit, the isotope label can be detected with high sensitivity during analysis. If the content of the isotope-labeled nutrient source is equal to or less than the above-mentioned upper limit, the concrete composition can be produced inexpensively.

[0026] The nutrient sources contained in the cement-containing molded body may all be isotope-labeled, but they do not all have to be isotope-labeled. For example, by using 0.01 mass% of the nutrient sources as isotope-labeled nutrient sources and multiplying the amount of isotope-labeled metabolites by 10,000, the amount of nutrient source decomposed by microorganisms can be estimated.

[0027] The concrete composition may contain chemical admixtures such as water reducers, viscosity improvers, and the like.

[0028] "First Step" In the first step, an acidic substance or a basic substance is applied to the surface of the cement-containing molded body. Examples of methods for applying the acidic substance or basic substance (application method) include a spray method in which a solution containing an acidic substance or a basic substance is sprayed onto the surface of the cement-containing molded body using a spray, a roll method in which the solution containing an acidic substance or a basic substance is applied to the surface of the cement-containing molded body using a roller, and a roll method in which the solution containing an acidic substance or a basic substance is applied to the surface of the cement-containing molded body using a brush.

[0029] The acidic substance is not particularly limited as long as it can dissolve the metabolic products produced when the microorganisms contained in the cement-containing molded body decompose the nutrient source, and examples thereof include hydrochloric acid, sulfuric acid, and nitric acid.

[0030] The concentration of the acidic substance is not particularly limited as long as it can dissolve the metabolic products generated when the microorganisms contained in the cement-containing molded body decompose the nutrient source, but for example, it is preferably 1% by mass or more, and more preferably 30% by mass or more. If the concentration of the acidic substance is equal to or higher than the above lower limit, the cement molded body can be sufficiently dissolved with ease.

[0031] The amount of the acidic substance applied to the surface of the cement-containing molded body is not particularly limited as long as it can dissolve the metabolic products generated when the microorganisms contained in the cement-containing molded body decompose the nutrient source. For example, the amount of the acidic substance applied to the surface of the cement-containing molded body is 10 g / m 2 More than 10000g / m2 Preferably, it is 50 g / m or less. 2 More than 1000g / m 2 It is more preferable that the amount of the acidic substance is equal to or greater than the above lower limit, a sufficient amount of metabolic products can be released. If the amount of the acidic substance is equal to or less than the above upper limit, changes in the shape and properties of the cement-containing molded body due to an excess of the acidic substance can be suppressed.

[0032] The basic substance is not particularly limited as long as it can dissolve metabolic products produced when the microorganisms contained in the cement-containing molded body decompose the nutrient source, and examples thereof include sodium hydroxide, calcium hydroxide, potassium hydroxide, etc. Specifically, a solution containing the basic substance is used. Examples of the solvent for the solution containing the basic substance include water and alcohol.

[0033] The concentration of the basic substance solution is not particularly limited as long as it can dissolve metabolic products generated when the microorganisms contained in the cement-containing molded body decompose the nutrient source, but is preferably 1% by mass or more, and more preferably 10% by mass or more. If the concentration of the basic substance solution is equal to or higher than the above-mentioned lower limit, the cement molded body can be sufficiently dissolved with ease.

[0034] The amount of the basic substance applied to the surface of the cement-containing molded body is not particularly limited as long as it can dissolve the metabolic products generated when the microorganisms contained in the cement-containing molded body decompose the nutrient source. For example, the amount of the basic substance applied to the surface of the cement-containing molded body is 10 g / m 2 More than 10000g / m 2 Preferably, it is 50 g / m or less. 2 More than 1000g / m 2 It is more preferable that the amount of the basic substance is equal to or greater than the above lower limit. If the amount of the basic substance is equal to or greater than the above upper limit, a sufficient amount of metabolic products can be released. If the amount of the basic substance is equal to or less than the above upper limit, changes in the shape and properties of the cement-containing molded body due to excess acidic substance can be suppressed.

[0035] By applying an acidic or basic substance to the surface of the cement-containing molded body in this way, metabolic products such as carbon dioxide (CO2) and ammonia (NH3) produced when the microorganisms contained in the cement-containing molded body decompose the nutrient source are released from the surface of the cement-containing molded body.

[0036] for example, 13 When C-labeled nutrients are used, the amount of carbon generated by microbial metabolism (the microorganisms consume nutrients and oxygen) 13 Most of the C-labeled CO2 precipitates as calcium carbonate (CaCO3) in the cement-containing compact. Therefore, by exposing the cement-containing compact to an acidic substance (e.g., hydrochloric acid), the CaCO3 dissolves in the acidic substance and releases CO2. 13 The amount of carbon is measured in the second step. 15 When N-labeled nutrients were used, the amount of microbial metabolism 15 Some of the N-labeled NH3 remains inside the cement-containing compact. Therefore, the cement-containing compact is exposed to a basic substance (e.g., sodium hydroxide) to dissolve the NH3 in the basic substance and release the NH3. 15 The amount of N is measured in a second step.

[0037] Examples of methods for recovering metabolic products (gases) released from the surface of a cement-containing molded body include: (1) a method in which one end of a tube connected to a suction device such as a vacuum pump is placed on the area on the surface of the cement-containing molded body where an acidic or basic substance has been applied, and the gas is recovered in a container using the suction device; and (2) a method in which a plastic bag is placed on the area on the surface of the cement-containing molded body where an acidic or basic substance has been applied, and the gas is recovered in the plastic bag.

[0038] "Second Step" In the second step, the amount of isotope contained in the metabolites recovered in the first step is measured to estimate the amount of isotope-labeled nutrient source decomposed by the microorganisms.

[0039] The amount of isotopes contained in metabolites is measured by mass spectrometry, which measures the amount of isotopes contained in the metabolite gas. The metabolite released as a gas is analyzed as a gas using a gas chromatograph mass spectrometer (GC-MS). The metabolite released as a gas is also recovered in a basic solution and analyzed as a liquid using a liquid chromatography mass spectrometer (LC-MS). In this way, the amount of the nutrient source decomposed by the microorganisms is estimated by measuring the amount of isotopes contained in the metabolites. As described above, at least a portion of the nutrient source contained in the cement-containing molded body is an isotope-labeled nutrient source. Therefore, the amount of the nutrient source actually decomposed by the microorganisms can be estimated from the amount of the isotope-labeled nutrient source decomposed by the microorganisms.

[0040] For example, carbonates are commonly present in cement-containing compacts because they are contained in the materials of the cement-containing compacts, or because carbonates are incorporated into the cement-containing compacts and the calcium in the cement-containing compacts reacts with CO2. 13 When C-labeled nutrients are not used, the carbonates that are ubiquitously present in cement-containing compacts and the microorganisms 13 It is not possible to distinguish between CO2 produced by the decomposition of C-labeled nutrients and CO2 produced by the decomposition of C-labeled nutrients. 13 When a C-labeled nutrient source is decomposed, CO2 with a mass number of 45 is generated. By analyzing the difference in mass number using a mass spectrometer, it is possible to detect CO2 even in an environment where various microorganisms exist. 13 Only the degradation of C-labeled nutrients can be specifically detected.

[0041] The method for measuring microbial activity in a cement-containing molded body of this embodiment includes the steps of applying an acidic or basic substance to the surface of the cement-containing molded body to release, from the surface of the cement-containing molded body, metabolic products produced when microorganisms contained in the cement-containing molded body decompose the isotope-labeled nutrient source, and recovering the metabolic products and measuring the amount of isotope contained in the metabolic products to estimate the amount of the isotope-labeled nutrient source decomposed by the microorganisms. Therefore, the method for measuring microbial activity in a cement-containing molded body of this embodiment can specifically and accurately estimate the activity of the microorganisms contained in the cement-containing molded body. Furthermore, by estimating the activity of the microorganisms contained in the cement-containing molded body, the repair state of self-healing concrete can be predicted as described below. The mechanism by which microorganisms repair concrete is thought to be as follows: When cracks occur in a cement-containing compact, oxygen and water penetrate into the compact, activating the microorganisms contained in the compact. Carbon dioxide generated by the metabolism of the microorganisms reacts with calcium in the compact to precipitate calcium carbonate, which fills the cracks in the compact. Therefore, if the amount of microbial activity can be estimated, it is possible to estimate how much calcium carbonate has been produced, i.e., how much concrete has been repaired. [Example]

[0042] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to the following examples.

[0043] [Example 1] A concrete composition containing polylactic acid was prepared as a nutrient source for microorganisms (bacteria). The concrete composition is shown in Table 1. As shown in Table 1, the concrete composition was 20 kg / m 3 The carbon content of polylactic acid is 40%, so this concrete composition has a carbon content of 8 kg / m 3 The concrete composition contains 0.1% of the polylactic acid.13 The concrete composition was 80 g / m 3 of 13 Contains C. The concrete composition was molded to obtain a cement-containing molded body. 30% by mass of hydrochloric acid was applied to the surface of the cement-containing molded body to dissolve the calcium carbonate contained in the cement-containing molded body and release carbon dioxide. The released carbon dioxide was collected in a container using a vacuum pump and analyzed by mass spectrometry using a gas chromatograph mass spectrometer (product name: GCMS-QP2020NX, manufactured by Shimadzu Corporation). 13 40 mg of C-labeled carbon dioxide was detected. As a result, it was found that 50% of the added nutrient source (polylactic acid) was decomposed by bacteria to carbon dioxide, which existed in the concrete as calcium carbonate. This method makes it possible to know how much polylactic acid contained in the cement-containing molded body has been consumed, and to estimate the amount of microbial activity. 3 Since 40g of carbon dioxide was generated by the metabolism of microorganisms in the cement-containing compact, 90g of calcium carbonate was generated by the metabolism of microorganisms in the cement-containing compact. 3 It can be estimated that self-repair equivalent to 90g of calcium carbonate occurred per unit area.

[0044] [Table 1]

[0045] [Example 2] A concrete composition containing urea as a nutrient source for microorganisms (bacteria) was prepared. The concrete composition is shown in Table 2. As shown in Table 2, the concrete composition was 20 kg / m 3 The carbon content of urea is 20%, so this concrete composition has a carbon content of 4 kg / m 3The concrete composition contains 0.1% of the polylactic acid. 13 The total concrete composition was 4 g / m 3 of 13 Contains C. The concrete composition was molded to obtain a cement-containing molded body. 30% by mass of hydrochloric acid was applied to the surface of the cement-containing molded body to dissolve the calcium carbonate contained in the cement-containing molded body and release carbon dioxide. The released carbon dioxide was collected in a container using a vacuum pump and subjected to mass analysis using the gas chromatograph mass spectrometer. 13 1 mg of C-labeled carbon dioxide was detected. As a result, it was found that 25% of the added nutrient source (urea) was decomposed by bacteria to carbon dioxide, which existed in the concrete as calcium carbonate. This method makes it possible to know how much urea contained in the cement-containing molded body has been consumed, and to estimate the amount of microbial activity. 3 Since 1 g of carbon dioxide was generated by the metabolism of microorganisms in the cement-containing compact, 2.3 g of calcium carbonate was generated by the metabolism of microorganisms in the cement-containing compact. 3 It can be estimated that self-repair occurred equivalent to 2.3 g of calcium carbonate per unit area.

[0046] [Table 2]

Claims

1. A method for measuring microbial activity in a cement-containing molded body obtained by molding a concrete composition containing a cement-containing composition, a microorganism, and a nutrient source, comprising: At least a portion of the nutrient source is an isotope-labeled nutrient source; a step of releasing metabolic products generated when the microorganisms contained in the cement-containing molded body decompose the isotope-labeled nutrient source from the surface of the cement-containing molded body, and recovering the metabolic products; and estimating the amount of the isotope-labeled nutrient source decomposed by the microorganism by measuring the amount of isotope contained in the metabolic product.

2. The isotope-labeled nutrient source is １３ C-labeled nutrient sources, １５ N-labeled nutrient sources and １７ 2. The method for measuring microbial activity in a cement-containing molded body according to claim 1, wherein the microbial activity is at least one selected from the group consisting of O-labeled nutrient sources.

3. 3. The method for measuring microbial activity in a cement-containing molded body according to claim 1, wherein the amount of isotope contained in the metabolite is measured by mass spectrometry of the metabolite.

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