Method for culturing hydrogen-oxidizing bacteria

Abstract

To provide a method for culturing hydrogen-oxidizing bacteria inexpensively and efficiently.SOLUTION: A method for culturing hydrogen-oxidizing bacteria comprises culturing hydrogen-oxidizing bacteria in a seawater medium in the presence of a material containing calcium silicate hydrate.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for culturing hydrogen-oxidizing bacteria. [Background technology]

[0002] In response to serious environmental issues such as global warming, efforts toward decarbonization are becoming more active around the world. Biological CO2 resource recovery is currently attracting attention as a technology with a low environmental impact, and representative examples include CO2 fixation by culturing microalgae and hydrogen-oxidizing bacteria. Hydrogen-oxidizing bacteria are characterized by their high proliferation rate and have excellent CO2 fixation ability among living organisms.

[0003] On the other hand, the creation of a hydrogen society through the utilization of hydrogen is considered to be important for realizing decarbonization, and the promotion of hydrogen utilization is expected to be emphasized even more in the future. Hydrogen-oxidizing bacteria have the potential to contribute to the important technological development of hydrogen utilization and CO2 resource utilization, and the utilization of hydrogen-oxidizing bacteria requires the establishment of a cheap and efficient cultivation method.

[0004] Conventionally, hydrogen-oxidizing bacteria are cultured in the presence of culture gases containing H2, O2, and CO2, at a salt concentration of 0-1 mol / L, and at a pH of around 5-8, using a medium containing many types of reagents such as sodium chloride, calcium chloride, and potassium dihydrogen phosphate, in order to appropriately promote proliferation (e.g., Patent Document 1, Non-Patent Document 1). Therefore, one of the problems is that the cost of reagents involved in the preparation of the medium is high, and the preparation requires accurate mixing, which is time-consuming and labor-intensive. In addition, as the culture of hydrogen-oxidizing bacteria proceeds, the pH in the culture system decreases due to acidic substances such as organic acids that they produce, and the culture system deviates from the pH range suitable for culturing hydrogen bacteria, so appropriate pH management is required. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Publication No. 5-115290 [Non-patent literature]

[0006] [Non-Patent Document 1] Journal of Fermentation and Bioengineering Vol.72, No.5, 358-361.1991 Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention relates to providing a method for culturing hydrogen-oxidizing bacteria inexpensively and efficiently. [Means for solving the problem]

[0008] The inventors have investigated the cultivation of hydrogen oxidizing bacteria and found that by adding a calcium silicate hydrate-containing material to the culture system, hydrogen oxidizing bacteria can be cultivated efficiently even when inexpensive seawater medium is used. That is, the present invention relates to the following 1) to 10). 1) A method for culturing hydrogen oxidizing bacteria, comprising culturing the hydrogen oxidizing bacteria in a seawater medium in the presence of a calcium silicate hydrate-containing material. 2) The method according to 1), wherein the seawater medium contains one or more sources selected from a nitrogen source, a phosphorus source, an iron source and a nickel source. 3) The method according to 1), wherein the salt concentration of the seawater medium is 0.2% by mass or more and 13% by mass or less. 4) The method according to any one of 1) to 3), wherein the hydrogen-oxidizing bacteria is a bacterium selected from the genera Hydrogenovibrio, Hydrogenobacter, Hydrogenophilus, Alcaligenes, Ralstonia, Cupriavidus, Hydrogenophaga, Pseudomonas, and Bacillus. 5) The method according to any one of 1) to 3), wherein the hydrogen-oxidizing bacteria are bacteria belonging to the genus Hydrogenovibrio or Hydrogenophilus. 6) The method according to any one of 1) to 3), wherein the hydrogen-oxidizing bacteria is Hydrogenovibrio marinus, Hydrogenovibrio crunogenus, Hydrogenovibrio halophilus, Hydrogenovibrio kuenenii, Hydrogenovibrio thermophilus, Hydrogenovibrio thermoluteolus, Hydrogenophilus denitrificans, Hydrogenophilus halorhabdus, or Hydrogenophilus hirschii. 7) Calcium silicate hydrate-containing materials have a density of 0.8 to 2.5 g / cm 3 The method according to any one of 1) to 6), wherein the porosity is 10 to 80%. 8) The method according to any one of 1) to 6), wherein the calcium silicate hydrate-containing material is a material containing one or more selected from the group consisting of tobermorite, xonotlite, CSH gel, foshagite, gyrolite, hillebrandite, and wollastonite. 9) The method according to any one of 1) to 8), wherein the calcium silicate hydrate-containing material is a calcium silicate hydrate-containing material that has been treated with phosphoric acid. 10) The method according to any one of 1) to 9), wherein the calcium silicate hydrate-containing material is used at a concentration in the medium of 0.1 g / L or more and 30 g / L or less in terms of calcium silicate hydrate. Effect of the Invention

[0009] According to the culture method of the present invention, the pH environment of the culture system can be stably maintained within a predetermined range, and a culture system suitable for culturing hydrogen-oxidizing bacteria can be maintained for a longer period of time. By using the method of the present invention, hydrogen-oxidizing bacteria can be efficiently cultured at low cost and with little labor. [Brief description of the drawings]

[0010] [Figure 1] 2 is a graph showing the change in bacterial cell concentration (OD value) when cultured in a seawater medium in the presence of a calcium silicate hydrate-containing material. [Diagram 2] 2 is a graph showing the change in bacterial cell concentration (OD value) when cultured in a seawater medium in the presence of a calcium silicate hydrate-containing material that has not been treated with phosphate. [Diagram 3] 2 is a graph showing the change in bacterial cell concentration (OD value) when cultured in a seawater medium in the absence of a calcium silicate hydrate-containing material. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] In the present invention, hydrogen-oxidizing bacteria is a general term for chemosynthetic bacteria that oxidize hydrogen and use the energy generated by the reaction to perform carbon assimilation. That is, hydrogen-oxidizing bacteria can take in hydrogen as an energy source and CO2 as a carbon source, and grow while converting CO2 into organic matter in the living body. Hydrogen-oxidizing bacteria are present in natural environments such as soil, oceans, and hot springs, and include bacteria belonging to the genera Hydrogenovibrio, Hydrogenobacter, Hydrogenophilus, Alcaligenes, Ralstonia, Cupriavidus, Hydrogenophaga, Pseudomonas, and Bacillus. In the present invention, however, bacteria belonging to the genera Hydrogenovibrio and Hydrogenophilus are preferred, and bacteria of the genus Hydrogenovibrio are more preferred.

[0012] Examples of the bacteria of the genus Hydrogenovibrio include Hydrogenovibrio marinus, Hydrogenovibrio crunogenus, Hydrogenovibrio halophilus, Hydrogenovibrio kuenenii, and Hydrogenovibrio thermophilus. Examples of the bacteria of the genus Hydrogenophilus include Hydrogenophilus thermoluteolus, Hydrogenophilus denitrificans, Hydrogenophilus halorhabdus, and Hydrogenophilus hirsi. hirschii) etc.

[0013] An example of a hydrogen-oxidizing bacterium suitable for the method of the present invention is Hydrogenovibrio marinus strain MH-110 (deposited at the RIKEN BioResource Research Center under the name JCM7688).

[0014] In the culture method of the present invention, a seawater medium is used as the medium. The "seawater" may be either natural seawater collected from the sea or artificial seawater artificially prepared to mimic the composition of seawater. Seawater may be diluted with fresh water, brackish water may be used instead of seawater, or seawater concentrated by evaporation, dialysis, or a seawater desalination device may be used. The salt concentration of seawater is not particularly limited, and seawater with a salt concentration of, for example, 0.2 to 10% by mass, preferably 0.35 to 8% by mass, may be used.

[0015] From the viewpoint of the growth of hydrogen-oxidizing bacteria, the medium preferably contains one or more of a nitrogen source, a phosphorus source, an iron source, and a nickel source, more preferably two or more, more preferably three or more, and further preferably contains all of a nitrogen source, a phosphorus source, an iron source, and a nickel source. Examples of the nitrogen source include ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium nitrate, and aqueous ammonia. The nitrogen source content, in terms of the ammonium species concentration in the medium, is preferably 1 mg-N / L or more, more preferably 10 mg-N / L or more, even more preferably 100 mg-N / L or more, and is preferably 4000 mg-N / L or less, more preferably 3500 mg-N / L or less, even more preferably 2500 mg-N / L or less.

[0016] Examples of phosphorus sources include phosphates such as dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and hydroxyapatite, and aqueous phosphoric acid solutions. The content of the phosphorus source, in terms of the concentration of phosphate species in the medium, is preferably 1 mg-P / L or more, more preferably 10 mg-P / L or more, even more preferably 100 mg-P / L or more, and preferably 2500 mg-P / L or less, more preferably 2000 mg-P / L or less, even more preferably 1000 mg-P / L or less.

[0017] Examples of iron sources include iron salts such as ferrous sulfate, ferric sulfate, and ferrous chloride, metallic iron capable of supplying iron ions in the medium, iron oxide, and compositions containing these. The content of the iron source, in terms of the soluble iron concentration in the medium, is preferably 0.01 mg-Fe / L or more, more preferably 0.1 mg-Fe / L or more, even more preferably 1 mg-Fe / L or more, and is preferably 250 mg-Fe / L or less, more preferably 200 mg-Fe / L or less, even more preferably 100 mg-Fe / L or less.

[0018] Examples of nickel sources include nickel salts such as nickel sulfate and nickel chloride, metallic nickel capable of supplying nickel ions in the medium, nickel oxide, nickel-containing alloys, and compositions containing these. The content of the nickel source, in terms of nickel ion concentration in the culture medium, is preferably 0.001 mg-Ni / L or more, more preferably 0.01 mg-Ni / L or more, even more preferably 0.1 mg-Ni / L or more, and is preferably 20 mg-Ni / L or less, more preferably 10 mg-Ni / L or less, even more preferably 5 mg-Ni / L or less.

[0019] The salt concentration of the seawater medium is preferably 0.2% by mass or more, more preferably 0.3% by mass or more, more preferably 0.35% by mass or more, and preferably 13% by mass or less, more preferably 12% by mass or less, more preferably 11% by mass or less.

[0020] In the culture method of the present invention, the culture is carried out in the presence of a calcium silicate hydrate-containing material, which makes it possible to maintain the pH environment of the culture system within a certain range without adversely affecting the growth of the bacteria, and thus makes it possible to maintain a culture system suitable for culturing hydrogen-oxidizing bacteria for a longer period of time. In the present invention, calcium silicate hydrate is a type of silicate, and is a general term for compositions in which calcium oxide, silicon dioxide and water are combined in various composition ratios. Calcium silicate hydrates can be classified according to their crystal structure, for example, into tobermorite, xonotlite, CSH gel, foshagite, gyrolite, hillebrandite, and wollastonite.

[0021] Tobermorite is a crystalline calcium silicate hydrate, e.g., Ca5·(SiO 18 H2)·4H2O (plate-like form), Ca5·(SiO 18 H2) (plate-like morphology), Ca5·(SiO 18 It has a chemical composition such as H2)·8H2O (fibrous form). Xonotlite is a crystalline calcium silicate hydrate, e.g., Ca6·(SiO 17 )·(OH)2 (fibrous form), etc. CSH gel has the chemical composition αCaO·βSiO2·γH2O (where α / β=0.7-2.3, γ / β=1.2-2.7). For example, calcium silicate hydrate has the chemical composition 3CaO·2SiO2·3H2O. Foshagite has the chemical composition Ca4(SiO3)3(OH)2 etc. Gyrolite is (NaCa2)Ca 14 (Si 23 Al)O 60 It has a chemical composition such as (OH)8·14H2O. Hillebrandite has the chemical composition Ca2SiO3(OH)2 etc. Wollastonite has the chemical composition of CaO·SiO2 (fibrous or columnar). Among these, tobermorite is preferred from the viewpoints of availability and economy.

[0022] In the present invention, the "calcium silicate hydrate-containing material" includes various materials and ingredients containing calcium silicate hydrate, such as calcium silicate hydrate itself, natural minerals, cement hydrate, and hardened cement bodies separated from waste concrete. From the viewpoint of easy availability, aerated lightweight concrete (ALC) containing tobermorite as a main component is preferable. Here, "lightweight aerated concrete" refers to a material consisting of tobermorite and unreacted silica stone. The proportion of tobermorite in the lightweight aerated concrete is usually about 50 to 80% by volume, assuming the entire solid phase excluding the voids inside the lightweight aerated concrete as 100% by volume. From the viewpoint of promoting the use of waste materials, scraps and waste materials of lightweight cellular concrete generated during the manufacturing process of lightweight cellular concrete or at construction sites may be used. Furthermore, scraps and waste materials of moisture retaining materials containing xonotlite may also be used.

[0023] Lightweight cellular concrete can be obtained, for example, by autoclaving raw materials (for example, a hardened body consisting of a mixture of these) containing silica powder, cement, quicklime powder, a foaming agent (for example, aluminum powder), water, etc.

[0024] The calcium silicate hydrate-containing material has a porosity of preferably 10 to 80%, more preferably 15 to 80%, and further preferably 20 to 75%. The term "porosity" refers to the ratio of the total volume of voids to the total volume of the support. The porosity can be measured by the mercury intrusion method shown in the examples below.

[0025] The calcium silicate hydrate-containing material preferably has a density of 0.8 to 2.5 g / cm 3 and more preferably 0.9 to 2.4 g / cm 3 and more preferably 1.0 to 2.3 g / cm 3 Here, the term "density" refers to the density of the material when mercury is injected into the pores by mercury intrusion porosimetry.

[0026] The calcium silicate hydrate-containing material may be in the form of either powder or granules, but from the viewpoint of quickly adjusting the pH, it is preferable to use a powder with a large surface area, and from the viewpoint of exerting the pH adjusting ability over a long period of time, it is preferable to use a granular form with a slower reaction rate than a powder. The powder form is not particularly limited, but for example, it can be a powder that passes through a sieve with a mesh size of 2 mm. The granular form is not particularly limited, but for example, it can be a granular form that passes through a sieve with a mesh size of 5 mm but does not pass through a sieve with a mesh size of 1 mm.

[0027] From the viewpoint of suppressing adsorption of phosphorus in the medium, the calcium silicate hydrate-containing material is preferably added to the medium in a phosphoric acid-treated state. The phosphoric acid treatment can be carried out, for example, by adding the calcium silicate hydrate-containing material to a 0.1 to 10 mass% aqueous phosphoric acid solution and stirring for about 1 second to 1 hour, followed by multiple washing operations using distilled water, and drying at 60 to 180°C.

[0028] The calcium silicate hydrate-containing material may be present in the culture system during the culture period, but it is preferable for it to be present in the culture system from the beginning of the culture, and it is preferable to add it to the culture medium before or simultaneously with inoculation of the hydrogen-oxidizing bacteria into the culture medium. The amount of calcium silicate hydrate-containing material used may be any amount that satisfies the pH buffering performance of the calcium silicate hydrate-containing material, specifically, that allows the pH of the medium to be adjusted to 3 to 11, more preferably 4 to 11, and even more preferably 5 to 10, and the concentration of calcium silicate hydrate in the medium is preferably 0.1 g / L or more, more preferably 0.5 g / L or more, more preferably 1 g / L or more, and is preferably 30 g / L or less, more preferably 25 g / L or less, and more preferably 20 g / L or less. When the calcium silicate hydrate-containing material is used without the above-mentioned phosphoric acid treatment, it is preferable to use it so that the concentration in the culture medium is 20 g / L or less, more preferably 15 g / L or less, in order to suppress the adsorption of phosphorus in the culture medium.

[0029] Cultivation is performed by inoculating a preculture solution of hydrogen-oxidizing bacteria into the above-mentioned seawater medium, and in the presence of a calcium silicate hydrate-containing material, in an atmosphere of H2, O2, and CO2, at 10 to 55°C, preferably 20 to 45°C, and at a pH of 3 to 11, preferably 4 to 10. The culture period is not particularly limited, but can be, for example, 0 to 48 hours, preferably 1 to 24 hours, when the culture is performed in a batch system.

[0030] The hydrogen-oxidizing bacteria can be pre-cultured, for example, by using a synthetic medium (pH 7) with a salt concentration similar to that of seawater, and culturing with shaking at 20 to 45°C for about 15 to 20 hours in an atmosphere of a mixed gas of, for example, H2:O2:CO2 = 7:2:1 (1 atm).

[0031] The pH can be appropriately adjusted within the range of pH 3 to 11 depending on the amount of calcium silicate hydrate-containing material used, but hydrogen chloride, sodium hydroxide, ammonia water, etc. may be used as necessary. The pH adjustment range is more preferably pH 4-10, and further preferably pH 5-9.

[0032] Thus, according to the culture method of the present invention, even when a seawater medium is used, the pH environment of the culture system can be maintained within a range suitable for the growth of hydrogen-oxidizing bacteria for a long period of time, and hydrogen-oxidizing bacteria can be efficiently cultured at low cost and with little labor, without adversely affecting the growth of the bacteria. EXAMPLES

[0033] 1. Hydrogen-oxidizing bacteria Hydrogenovibrio marinus MH-110 was cultured as follows to prepare a preculture, which was then used in the following cultivation. The Hydrogenovibrio marinus MH-110 strain was obtained from the RIKEN BioResource Research Center (JCM7688) and used. <Preculture> 1) A synthetic medium (pH 7) with the following composition (content of ingredients per 1 L) and a salt concentration similar to that of seawater was used: 5.0g (NH4)2SO4,2.5g K2HPO4,0.5g KH2PO4,0.5g MgSO4·7H2O,29.3g NaCl,10mg CaCl2·2H2O,10mg FeSO4·7H2O,0.6mg NiSO4·6H2O,2.0ml Trace element solution. The trace element solution had the following composition (contents per liter): 1.0mg MoO3,7.0mg ZnSO4·7H2O,0.5mg CuSO4·5H2O,1.0mg H3BO3,1.0mg MnSO4·5H2O,1.0mg CoCl2·6H2O. 2) 150 mL of synthetic medium was placed in a 1 L Sakaguchi flask, and the gas phase was replaced with a mixed gas of H2:O2:CO2 = 7:2:1 (1 atm) and cultured with shaking at 30°C. After 15 hours of culture, the gas phase was replaced with the above mixed gas and cultured for another 3 hours. The mixed gas was adjusted to an arbitrary composition by controlling the flow rate of each component gas with a mass flow controller (HORIBA CU-2130 H2, O2, CO2).

[0034] 2. Calcium silicate hydrate-containing materials (1) Calcium silicate hydrate-containing materials In this example, lightweight cellular concrete was crushed and sieved to obtain powders with particle sizes of 1 to 4 mm and 1 mm or less. 3 The porosity was 57%. For materials with a particle size of 1 mm or less (powdered materials), the density was 1.59 g / cm 3 , and the porosity was 66%. The density and porosity were measured by mercury intrusion porosimetry using an AutoPore IV 9520 (Micromeritics, Shimadzu Corporation). The density A of the material was measured when the force of mercury being forced into the pores was 0.49 pounds per square inch and when no mercury was forced into the pores. In addition, the density B of the material was measured when mercury was forced into the pores at a force of 59,800 pounds per square inch. Here, the "density" refers to the density of the material when mercury is forced into the pores by mercury intrusion porosimetry. "Porosity" refers to the ratio of the total volume of voids to the total volume of a material. Porosity can be measured by mercury intrusion porosimetry. The porosity is calculated according to the following (Equation 1). (Formula 1) Porosity (%)=100×[1-(A / B)] The symbols in the formula are as follows. A: Density of the material measured by mercury intrusion porosimetry without mercury being intruded into the pores B: Density of the material measured by mercury intrusion porosimetry with mercury injecting into the pores

[0035] (2) Phosphate treatment 10 g of the calcium silicate hydrate-containing material (1) was added to 200 ml of 0.85% phosphoric acid aqueous solution. After stirring for 5 seconds, the phosphoric acid aqueous solution was completely discarded. As a washing operation, 100 ml of distilled water was added, stirred for 5 seconds, and the washing liquid was discarded, which was repeated three times. The treated calcium silicate hydrate-containing material was dried overnight in a dryer set at 105°C.

[0036] (3) Evaluation of the pH buffering performance of calcium silicate hydrate-containing materials treated with phosphoric acid and calcium silicate hydrate-containing materials not treated with phosphoric acid (determination of the amount to be used) The phosphoric acid-treated calcium silicate hydrate-containing material prepared in (2) and the untreated calcium silicate hydrate-containing material were added to 200 ml of distilled water to a concentration of 0.1 g / L and 1 g / L, and stirred. After stirring for 20 minutes and measuring the pH of the solution, 0.5 ml of a nitric acid aqueous solution with a concentration of 1% by mass was added and stirred again. 20 minutes after the addition of the nitric acid aqueous solution, 0.5 ml of a nitric acid aqueous solution with a concentration of 1% by mass was added and stirred again. By repeating this procedure, the pH buffering performance due to the addition of the phosphoric acid-treated calcium silicate hydrate-containing material and the untreated calcium silicate hydrate-containing material was evaluated (Tables 1 and 2 below). From this evaluation, the pH adjustment speed and the strength of the adjustment action can be evaluated. From the results in Table 1, it is clear that the use of untreated calcium silicate hydrate-containing material in powder form at a concentration of 0.1 g / L or more, or in granular form at a concentration of 1 g / L or more, will enable the material to exhibit pH buffering properties in a short period of time. From the results in Table 2, it is clear that the phosphoric acid-treated calcium silicate hydrate-containing material exhibits pH buffering performance in a short time when used in powder form at a concentration of 1 g / L or more. In addition, although immediate effect was not confirmed, it was confirmed that the phosphoric acid-treated calcium silicate hydrate-containing material, even in granular form, can exhibit pH buffering performance when used at a concentration of 1 g / L or more.

[0037] [Table 1]

[0038] [Table 2]

[0039] (4) The following procedure was used to evaluate whether a calcium silicate hydrate-containing material that had been treated with phosphoric acid and a calcium silicate hydrate-containing material that had not been treated with phosphoric acid exhibited pH buffering performance when allowed to react for a sufficient period of time. The calcium silicate hydrate-containing material treated with phosphoric acid prepared in (2) and the calcium silicate hydrate-containing material not treated with phosphoric acid were added to distilled water to a concentration of 0.1 g / L and 1 g / L, and then placed in a sealed container together with a specified amount of 1 mass percent nitric acid and shaken for 16 hours (Table 3). From these results, it was confirmed that even the calcium silicate hydrate-containing material, for which pH buffering ability was not confirmed in the evaluation (3), exhibits acid buffering ability. That is, regardless of the particle size or the presence or absence of phosphoric acid treatment, the calcium silicate hydrate-containing material can have a pH buffering effect by using it at a concentration of 0.1 g / L or more. In addition, by increasing the concentration of the calcium silicate hydrate-containing material, the pH buffering effect can be exerted even for larger amounts or higher concentrations of acid.

[0040] [Table 3]

[0041] Example 1 (1) Preparation of seawater medium N, P, Fe, and Ni sources were added to a 250 ml jar fermenter (manufactured by Able Co., Ltd.) to prepare a seawater medium having the composition shown in Table 4 below.

[0042] [Table 4]

[0043] (2)Culture 13 ml of preculture of hydrogen oxidizing bacteria Hydrogenovibrio marinus MH-110 was inoculated into 140 ml of this seawater medium. Then, the medium was stirred at 800 rpm, and a gas mixture of H2:O2:CO2 gas in a volume ratio of 8:1:1 was aerated into the medium at a flow rate of 200 ml / min. After the start of the culture, Hm-KP buffer (phosphate component) consumed with the growth of hydrogen oxidizing bacteria was appropriately added, and a calcium silicate hydrate-containing material that was phosphoric acid-treated at the start of the culture was added to a concentration of 1 g / L, and an alkaline solution (1 mol / L sodium hydroxide aqueous solution, 4% ammonia water) was appropriately added to maintain the pH at 6.5, and the culture was performed at 34°C for 9 hours. OD (Optical Density, 540 nm) was measured as an index of the amount of microorganisms.

[0044] (3) Results The results are shown in Figure 1. As shown in Figure 1, during the cultivation period, a logarithmic growth phase with a fast growth rate was maintained, with the OD540 value exceeding 1 three hours after the start of cultivation. It is clear that the combined use of calcium silicate hydrate-containing material with pH buffering properties prevented excessive addition of alkaline solution for pH adjustment, and cultivation conditions in which the microorganisms could grow actively were maintained. In addition, high-density cultivation with an OD540 of over 10 was achieved, confirming that the calcium silicate hydrate-containing material itself does not inhibit microbial growth and contributes to excellent bacterial biomass productivity.

[0045] Example 2 At the start of the culture, a calcium silicate hydrate-containing material that had not been treated with phosphoric acid was added to a concentration of 0.5 g / L, and culture was carried out under the same conditions as in Example 1 for 6 hours.

[0046] The results of Example 2 are shown in FIG. 2, as in Example 1, a logarithmic growth phase with a fast growth rate is confirmed, with the OD540 value exceeding 1 3 hours after the start of culture. It is therefore clear that culture conditions in which microorganisms can grow actively have been obtained by the combined use of a calcium silicate hydrate-containing material with pH buffering properties.

[0047] Comparative Example 1 15 ml of preculture of hydrogen oxidizing bacteria Hydrogenovibrio marinus MH-110 was inoculated into 160 ml of seawater medium with the composition shown in Table 4. As in Example 1(2), the medium was stirred at 800 rpm, and a gas mixture of H2:O2:CO2 gas in a volume ratio of 8:1:1 was aerated into the medium at a flow rate of 200 ml / min. After starting the cultivation of hydrogen oxidizing bacteria in this way, the pH of the culture solution was maintained at 6.5 by appropriately adding Hm-KP buffer (phosphate component) that is consumed with the growth, as well as alkaline solution (1 mol / L sodium hydroxide aqueous solution, 4% ammonia water). The cultivation temperature was 34°C for 9 hours, and OD (540 nm) was measured as an indicator of the amount of microorganisms.

[0048] The results are shown in Figure 3. From Fig. 3, it can be seen that when no calcium silicate hydrate-containing material was added, the OD540 value did not reach 1 at 3 hours after the start of the culture, indicating a slower growth rate than in Examples 1 and 2. In addition, as the culture progressed, a larger amount of alkaline solution was added to maintain the pH at 6.5, causing growth inhibition. As a result, the growth of the bacteria stopped when the OD540 value reached 6.55, and it was confirmed that the lysis of the cells began and the OD540 value decreased. From this result, it is clear that the addition of an excessive amount of alkaline solution created conditions in which the microorganisms could not grow, and the resulting microbial concentration decreased. Therefore, under the culture conditions of Comparative Example 1, which does not use a calcium silicate hydrate-containing material, it is not possible to stably culture hydrogen-oxidizing bacteria at high speed and at high density using a seawater medium.

Claims

1. A method for culturing hydrogen oxidizing bacteria, comprising culturing the hydrogen oxidizing bacteria in a seawater medium in the presence of a calcium silicate hydrate-containing material.

2. The method according to claim 1, wherein the seawater medium contains one or more sources selected from a nitrogen source, a phosphorus source, an iron source and a nickel source.

3. The method according to claim 1, wherein the salt concentration of the seawater medium is 0.2% by mass or more and 13% by mass or less.

4. 2. The method according to claim 1, wherein the hydrogen-oxidizing bacteria is a bacterium selected from the genera Hydrogenovibrio, Hydrogenobacter, Hydrogenophilus, Alcaligenes, Ralstonia, Cupriavidus, Hydrogenophaga, Pseudomonas and Bacillus.

5. The method according to claim 1 , wherein the hydrogen-oxidizing bacteria are bacteria belonging to the genus Hydrogenovibrio or Hydrogenophilus.

6. Hydrogen-oxidizing bacteria include Hydrogenovibrio marinus, Hydrogenovibrio crunogenus, Hydrogenovibrio halophilus, Hydrogenovibrio kuenenii, Hydrogenovibrio thermophilus, Hydrogenophilus thermoluteolus, Hydrogenophilus denitrificans, Hydrogenovibrio marinus, Hydrogenovibrio crunogenus, Hydrogenovibrio halophilus, Hydrogenovibrio kuenenii, Hydrogenovibrio thermophilus, Hydrogenovibrio ... thermoluteolus, Hydrogenophilus denitrificans, Hydrogenovibrio marinus, Hydrogenovibrio crunogenus, Hydrogenovibrio halophilus, Hydrogenovibrio kuenenii, Hydrogenovibrio thermophilus, Hydrogenovibrio thermoluteolus, Hydrogenovibrio halophilus, Hydrogenovibrio kuenenii, Hydrogenovibrio thermophilus, Hydrogenovibrio thermoluteolus, Hydrogenovibrio thermoluteolus, Hydrogenovibrio thermoluteolus, Hydrogenovibrio thermoluteolus, Hydrogenovibrio thermoluteolus, Hydrogenovibrio thermoluteolus, Hydrogenovibrio thermoluteolus, Hydrogenovibrio thermoluteolus, Hydrogenovibrio thermoluteolus, Hydrogenovibrio thermoluteolus, Hydrogenovibrio thermoluteolus, Hydrogenovibrio thermoluteolus, Hydrogenovibrio thermoluteolus, Hydrogenovib 2. The method according to claim 1, wherein the host organism is Hydrogenophilus halorhabdus or Hydrogenophilus hirschii.

7. The calcium silicate hydrate-containing material has a density of 0.8 to 2.5 g / cm ３ The method according to any one of claims 1 to 6, wherein the porosity is from 10 to 80%.

8. The method according to any one of claims 1 to 6, wherein the calcium silicate hydrate-containing material is a material containing at least one selected from the group consisting of tobermorite, xonotlite, CSH gel, foshagite, gyrolite, hillebrandite and wollastonite.

9. The method according to any one of claims 1 to 6, wherein the calcium silicate hydrate containing material is a phosphate-treated calcium silicate hydrate containing material.

10. The method according to any one of claims 1 to 6, wherein the calcium silicate hydrate-containing material is used in a culture medium having a concentration of 0.1 g / L or more and 30 g / L or less in terms of calcium silicate hydrate.

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