JP2008061521A - Method for producing hydrogen, and apparatus for producing hydrogen - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing hydrogen at high hydrogen production rate and in improved productivity, comprising culturing of a hydrogen-producing anaerobic hyperthermophilic microorganism, such as Thermococcus kodakaraensis 12 at high density, and to provide an apparatus for producing hydrogen that is suitable for the method for producing hydrogen. <P>SOLUTION: This method for producing hydrogen carries out culturing of hydrogen-producing anaerobic hyperthermophilic microorganisms 12 by using a support 11 and a culture solution containing an amino acid source and a carbon source. This apparatus for producing the hydrogen comprises a culture tank 10, in which the support 11 and the hydrogen-producing anaerobic hyperthermophilic microorganisms 12 are charged, a culture solution-supply means 20 for supplying the culture solution, containing the amino acid source and the carbon source to the culture tank 10, a waste solution recovering means 30 for recovering the waste solution from the culture tank, and a hydrogen gas recovering means 40 for recovering hydrogen that is generated inside the culture tank. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a hydrogen production method and hydrogen production apparatus using hydrogen-producing anaerobic hyperthermophilic bacteria.

Among the anaerobic hyperthermophilic bacteria are hydrogen-producing anaerobic hyperthermophilic bacteria that have the property of producing hydrogen during cell division. An example of such a hydrogen-producing anaerobic hyperthermophilic bacterium is Thermococcus kodakaraensis. A technique for biologically producing hydrogen by culturing Tk has been conventionally proposed (see, for example, Patent Document 1). Patent Document 1 describes a method for producing hydrogen in which a microorganism is produced by culturing a microorganism of the genus Thermococcus. Moreover, it is described that the microorganism of the genus Thermococcus is Thermococcus kodakaraensis. Moreover, it describes that the said culture | cultivation is performed at the temperature of 60-105 degreeC in the absence of anaerobic atmosphere and elemental sulfur, without light irradiation.
In addition, although the microorganism name should be expressed in italics, it is displayed in normal font in this specification. In the present invention, “hydrogen-producing anaerobic hyperthermophilic bacterium” is defined as an anaerobic hyperthermophilic bacterium having a property of producing hydrogen in the process of cell division. The details of Thermococcus kodakaraensis are described in Non-Patent Documents 1 to 5.

  In addition, hydrogen supply equipment using hydrogen-producing anaerobic hyperthermophilic bacteria other than Thermococcus kodakaraensis has been proposed (see, for example, Patent Document 2). This Patent Document 2 discloses a culture apparatus for culturing hydrogen-producing anaerobic hyperthermophilic bacteria using an organic substance as a nutrient source in an anaerobic atmosphere at a temperature higher than normal temperature, and an exhaust heat holding medium of 80 ° C. or higher discharged from the exhaust heat source. A culture temperature maintaining mechanism that is supplied with heat to maintain the culture apparatus at a temperature suitable for the growth of the hydrogen-producing anaerobic hyperthermophilic bacteria, and the organic matter necessary for the growth of the hydrogen-producing anaerobic hyperthermophilic bacteria. An organic substance supply mechanism for supplying the organic substance into the culture apparatus; and a hydrogen storage unit for extracting and storing hydrogen generated in the culture apparatus by the growth of the hydrogen-producing anaerobic hyperthermophile from the culture apparatus. A hydrogen supply facility configured to be able to take out hydrogen from a reservoir is described. Further, it is described that the hydrogen-producing anaerobic hyperthermophilic bacterium is Pyrococcus furiosus or Thermotoga maritima, and the temperature suitable for the growth is 80 to 103 ° C.

On the other hand, in methane fermentation by methane bacteria, a method of increasing the cell density and speeding up methane fermentation by filling ceramic or plastic inside the reaction tank containing methane bacteria and culturing methane bacteria on the surface (Anaerobic fixed bed method) has been proposed (see, for example, Non-Patent Document 6). In addition, the granule (granular sludge) is prepared using the self-aggregation action of methane bacteria, and this granule is filled in the reaction tank to increase the density of methane bacteria, and then an upflow anaerobic reaction is performed for methane fermentation. Upflow Anaerobic Sludge Blanket (UASB) method has been put into practical use.
JP 2003-116589 A Japanese Patent Application Laid-Open No. 8-308587 "Description of Thermococcus kodakaraensis sp. Nov., A well studied hyperthermophilic archaeon previously reported as Pyrococcus sp. KOD1", H. Atomi, T. Fukui, T. Kanai, M. Morikawa, and T. Imanaka, Archaea, 1, 263 -267 (2004) "Complete genome sequence of the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 and comparison with Pyrococcus genomes", T. Fukui, H. Atomi, T. Kanai, R. Matsumi, S. Fujiwara, and T. Imanaka, Genome Res., 15 (3 ): 352-363 (2005) "Characterization of a cytosolic NiFe-hydrogenase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1", T. Kanai, S. Ito, and T. Imanaka, J. Bacteriol., 185, 1705-1711 (2003) "Targeted gene disruption by homologous recombination in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1", T. Sato, T. Fukui, H. Atomi, and T. Imanaka, J. Bacteriol., 185, 210-220 (2003) "Continuous hydrogen production by the hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1", T. Kanai, H. Imanaka, A. Nakajima, K. Uwamori, Y. Omori, T. Fukui, H. Atomi, and T. Imanaka, J. Biotechnol ., 116 (3): 271-282 (2005) "Waste Bioconversion-Organic Waste Recycling", Authors: Mieko Yada, Hiroko Kawaguchi, Ken Sasaki, Publication Date: June 15, 1996 (First Edition, First Print), Publisher: Kamijo, Reference page: 82-89

  However, the hydrogen production method according to Patent Document 1 and the hydrogen supply facility according to Patent Document 2 have a problem that the hydrogen production rate is slow and the hydrogen productivity is low. Further, Non-Patent Document 1 does not describe the applicability of Thermococcus kodakaraensis to the anaerobic fixed bed method, and thermococcus kodakaraensis has no self-aggregation action, so its high density There has been a problem that the method has not been established.

  The present invention provides a method for producing hydrogen in which hydrogen production rate and productivity are improved by high-density culture of a hydrogen-producing anaerobic hyperthermophilic bacterium such as Thermococcus kodakaraensis in view of the above-mentioned problems of the prior art. For the purpose. It is another object of the present invention to provide a hydrogen production apparatus suitable for this hydrogen production method.

To solve this problem,
A first aspect of the present invention is a method for producing hydrogen by producing hydrogen by culturing a hydrogen-producing anaerobic hyperthermophilic bacterium using a support and a culture solution containing an amino acid source and a carbon source. is there. Specifically, a step of supplying a support, a hydrogen-producing anaerobic hyperthermophilic bacterium, and a culture solution containing an amino acid source and a carbon source into the culture vessel, and a hydrogen-producing anaerobic hyperphilic organism in the culture solution. And a step of producing hydrogen by culturing thermophilic bacteria.

  In the hydrogen production method of the present invention, the support is preferably porous. The support is made of natural sponge, synthetic sponge, activated carbon, ceramic, ion exchange resin, zeolite, foamed stone, molten slag, cellulose acetate fiber, glass beads, carbon, silica gel, sand, alumina, molecular sieve, and titanium. One or more selected from the group consisting of oxides are preferred. Moreover, it is preferable that the said carbon source is 1 type (s) or 2 or more types chosen from the group which consists of pyruvic acid, a birubate, and a starch-type polysaccharide. The hydrogen-producing anaerobic hyperthermophilic bacterium is preferably Thermococcus kodakaraensis. The thermococcus kodakaraensis is a thermococcus kodakaraensis KOD1 (deposit organization: RIKEN Bioresource Center, Microbial Materials Development Office, deposit number: JCM 12380; or depository organization: American Type Culture Collection ( ATCC), accession number: ATCC BAA-918).

  According to a second aspect of the present invention, there is provided a culture vessel filled with a support and a hydrogen-producing anaerobic hyperthermophilic bacterium, and a culture solution supply means for supplying a culture solution containing an amino acid source and a carbon source to the culture vessel. A hydrogen production apparatus comprising waste liquid recovery means for recovering waste liquid from the culture tank and hydrogen gas recovery means for recovering hydrogen gas generated in the culture tank.

  In the hydrogen production apparatus of the present invention, it is preferable to further include an alkaline solution supply means for supplying an alkaline solution to the culture tank. The support is preferably porous. The support is made of natural sponge, synthetic sponge, activated carbon, ceramic, ion exchange resin, zeolite, foamed stone, molten slag, cellulose acetate fiber, glass beads, carbon, silica gel, sand, alumina, molecular sieve, and titanium. One or more selected from the group consisting of oxides are preferred. Moreover, it is preferable that the said carbon source is 1 type (s) or 2 or more types chosen from the group which consists of pyruvic acid, a birubate, and a starch-type polysaccharide. The hydrogen-producing anaerobic hyperthermophilic bacterium is preferably Thermococcus kodakaraensis. The Thermococcus kodakaraensis is preferably Thermococcus kodakaraensis KOD1.

  According to the present invention, since the hydrogen-producing anaerobic hyperthermophilic bacteria such as Thermococcus kodakaraensis are adsorbed to the support and cultured, the immobilization and densification of the hydrogen-producing anaerobic hyperthermophilic bacteria can be achieved. Can be easily achieved. Moreover, the production rate and productivity of hydrogen can be improved by producing hydrogen using a densified hydrogen-producing anaerobic hyperthermophilic bacterium.

  Hereinafter, an embodiment of the present invention using Thermococcus kodakaraensis as a hydrogen-producing anaerobic hyperthermophilic bacterium will be described in detail.

(Method for producing hydrogen)
Thermococcus kodakaraensis (hereinafter sometimes simply referred to as “bacteria”) used in the present invention has the property of producing hydrogen while cell division, as described above. Therefore, the increase in the bacterial cell concentration is proportional to the hydrogen production amount. In addition, bacterial cells excrete polysaccharides when they metabolize carbon sources. This polysaccharide is sticky, and has a function of capturing and immobilizing bacterial cells when a predetermined amount is aggregated. The thus formed polysaccharide and bacterial aggregates are generally called biofilms.
In addition to the above, Thermococcus kodakaraensis has the following characteristics 1) to 4).
1) Since the culture temperature is 60 to 105 ° C., contamination due to the propagation of other bacteria can be avoided.
2) Hydrogen production can be controlled by temperature change.
3) When organic waste is used as a nutrient source, solubilization of organic waste (especially starch-based polysaccharides) and hydrogen production reaction can proceed simultaneously.
4) When the produced hydrogen is used as the hydrogen source of the fuel cell, the generated electromotive force is high due to the high-temperature fermented hydrogen production.

Therefore, in the method for producing hydrogen of the present invention, first, a support, Thermococcus kodakaraensis, and a culture solution containing an amino acid source and a carbon source are supplied into the culture tank.
Examples of Thermococcus kodakaraensis include Thermococcus kodakaraensis KOD1 (hereinafter abbreviated as “KOD1 strain”), and genetic recombinants and mutants of KOD1 strain, both of which are suitable targets of the present invention. It is. Of these, the KOD1 strain is particularly suitable. The reason is described below.

  The KOD1 strain is registered with the Depositary Organization: RIKEN BioResource Center, Microbial Materials Development Office (Accession Number: JCM 12380), or the Depository Organization: American Type Culture Collection (ATCC) (Accession Number: ATCC BAA-918). Since it is deposited, it is easy to obtain. Further, the entire base sequence of the KOD1 strain has been determined, and gene recombination is easy to perform. Further, it has been found that the cell extract of the KOD1 strain has a high hydrogenase (biocatalyst involved in hydrogen production) activity. Moreover, since KOD1 strain can utilize starch as a carbon source, organic waste can be used for the culture solution. Moreover, the culture conditions and hydrogen production conditions of the KOD1 strain have already been clarified. In addition, since the KOD1 strain is a wild strain, its growth ability is stable.

  Some yeasts and various fungi have a self-aggregation property, and even if left undisturbed, they spontaneously agglomerate. However, the fungus bodies targeted by the present invention do not have a self-aggregation property. Therefore, in order to generate hydrogen efficiently, it is necessary to artificially realize self-aggregation. This is a fact found by the inventors through various studies. Then, a support is used as a means for realizing the self-aggregation property in a pseudo manner. The support adds a self-aggregating action (that is, a biofilm forming action) to the bacterial cells.

  Specifically, the support is one that adsorbs bacterial cells. This support is preferably porous. The distribution of pore diameters in the porous support is not constant, and most of them have a large statistical dispersion. On the other hand, the cell size is about 1 μm when the KOD1 strain matures, for example. Therefore, the pore diameter of the porous support is preferably 1 μm or more. This makes it easier for the bacterial cells to enter the porous support and be adsorbed.

  Further, the pore diameter of the porous support is more preferably 20 μm or more. Thereby, the proliferated microbial cells can be held inside the porous support. As a result, the cell density can be increased. The pore diameter of the porous support is more preferably 100 μm or more. Thereby, while supplying a culture solution efficiently to the microbial cells adsorbed inside the porous support, the consumed culture solution (waste solution) can be efficiently discharged to the outside of the porous support. it can. As a result, it is possible to promote the growth of microbial cells in the porous support body. In addition, since a biofilm can be formed inside the porous support, the cells in the porous support can be more immobilized.

In addition, it is preferable that the hole diameter of the hole of a porous support body is 300 micrometers or less. Thereby, the surface area of a porous support body can be made sufficiently wide, and the adsorption amount of a microbial cell can be increased.
The shape of the support is not particularly limited, but the volume is preferably small. When the volume is small, when a predetermined amount of the support is filled in the culture tank, the surface area of the entire support increases with respect to the volume of the culture tank, making it easier to immobilize the cells and forming a biofilm with the support as the core. can do.

The support is preferably subjected to adsorbent coating or surface treatment. Thereby, the adsorption amount of a microbial cell can further be increased. As this adsorbent and surface treatment, those that adsorb microorganisms and are stable at the culture temperature of the cells are preferably used.
For example, there is a cellulose carrier (trade name: Cellsnow ^™ ) manufactured by Kirin Beer / Sake Engineering Co., Ltd.

  The material of the support only needs to have a function of adsorbing bacterial cells, for example, natural sponge, synthetic sponge, activated carbon, ceramic, ion exchange resin, zeolite, foam stone, molten slag, cellulose acetate fiber, glass beads, It is preferably one or more selected from the group consisting of carbon, silica gel, sand, alumina, molecular sieve, and titanium oxide. Since these supports are easily available and easy to process, they can be suitably used in the present invention.

The culture solution used in the present invention contains an amino acid source and a carbon source. This amino acid source may be any one that is generally used for culturing bacterial cells.
Examples of the carbon source include pyruvic acid, pyruvate, and starch-based polysaccharides. These carbon sources are used alone or in combination.

Since pyruvic acid is acidic, when this pyruvic acid is used at a high concentration, the pH of the culture solution decreases. Therefore, when pyruvic acid is used as a carbon source at a high concentration, it is preferable to adjust the pH of the culture solution to 5 to 9 using a pH adjuster. Thereby, a microbial cell can be cultured favorably.
In addition, when pyruvate is used instead of pyruvic acid, the pH of the culture solution does not decrease, so there is no need to use a pH adjuster. Examples of the pyruvate include sodium pyruvate. This sodium pyruvate can be produced by a fermentation method using glucose as a raw material. It can also be produced by chemical synthesis.

  As starch-based polysaccharides, raw starch such as corn starch, potato starch, sweet potato starch, wheat starch, cassava starch, sago starch, tapioca starch, rice starch, bean starch, waste starch, warabi starch, lotus starch, diamond starch; Physically modified starch such as α-starch, fractionated amylose, wet heat-treated starch; hydrolyzed dextrin, enzymatically modified dextrin, enzyme modified starch such as amylose; chemically degraded starch; chemically modified starch and the like.

  The starch-based polysaccharide is preferably soluble when added to the culture solution, but is not particularly limited because it is easily solubilized by heating with water. When culturing using a hyperthermophilic bacterium, since it is often cultured at a high temperature of about 85 ° C., there is an advantage that even starch that is insoluble at room temperature can be easily solubilized. As is well known, starch is contained in a large amount in seeds, rhizomes and the like of higher plants as storage carbohydrates.

  The concentration of pyruvic acid, pyruvate, and starch-based polysaccharide in the culture solution may be any value that is suitable for culturing cells, and includes, for example, a range of 0.01 to 20% by weight. The pyruvate concentration is calculated by ignoring the weight of the base.

In addition to the above-mentioned amino acid source and carbon source, additives such as an antifoaming agent that stabilizes the liquid level of the culture solution in the culture tank and a dry yeast extract that is a vitamin source are added to the culture solution as necessary. It is preferable to do. In addition, when supplying a culture solution in a culture tank, the dissolved oxygen in a culture solution is substituted by inert gas, such as nitrogen and argon, beforehand. Thereby, it can prevent that culture | cultivation is inhibited by oxygen.
In addition, although the culture solution mentioned above may prepare the said component artificially, you may utilize the factory wastewater or domestic wastewater with abundant amino acid sources and carbon sources as a raw material of a culture solution. Thereby, waste processing can be achieved simultaneously with preparation of a culture solution.

As a method for introducing the support, the fungus body, and the culture solution into the culture tank, known methods can be used. For example, a culture solution that has been sterilized in advance may be supplied into the culture tank, and then the support and the cells may be added to the culture tank. Moreover, after supplying a support body and a culture solution in a culture tank and performing a sterilization process, you may add a microbial cell to a culture solution. Further, the cells are inoculated into a culture solution and pre-cultured, and the pre-cultured culture solution is added to a culture tank having a sterilized support and a culture solution to start main culture. May be.
In addition, when introduce | transducing a microbial cell in a culture tank, it is preferable to replace the air in a culture tank with an inert gas previously. Examples of the inert gas include nitrogen or argon. Thereby, it can prevent that culture | cultivation is inhibited by oxygen.

  In this way, the cells are cultured in the culture solution. The culture temperature should just be a value suitable for culture | cultivation of a microbial cell, for example, 60-105 degreeC is mentioned. The optimum growth temperature of Thermococcus kodakaraensis is about 85 ° C., and it is preferable that the temperature is closer to this temperature. Moreover, the pH of a culture solution should just be a value suitable for culture | cultivation of a microbial cell, for example, 6.0-7.0 are mentioned. Since the optimal growth pH of Thermococcus kodakaraensis is around 6.4, the closer to this pH value, the better.

As the culture progresses, the bacterial cell concentration in the culture tank increases and the demand for nutrients also increases. Therefore, it is preferable to adjust the amount of the culture solution in the culture tank, the supply rate of the culture solution, and the medium composition concentration of the supplied culture solution according to the bacterial cell concentration. Thereby, the growth rate of a microbial cell can be hold | maintained optimally.
In addition, as described in Examples described later, the concentration of the carbon source and the amino acid source is proportional to the bacterial cell concentration. Therefore, it is preferable to adjust the carbon source and amino acid source among the components of the medium composition according to the bacterial cell concentration.

  When the cells are cultured in the presence of a support, they are adsorbed on the support and proliferate while excreting the polysaccharide derived from the carbon source. As a result, a biofilm is formed by the polysaccharides aggregating on the support and taking up the cells. Biofilms have chemical properties that facilitate the adsorption of bacterial cells, and the bacterial cells are further cultured in the biofilm. As a result, fixation and densification of the bacterial cells are achieved.

  Thus, since the microbial cells are fixed with high density in the biofilm, the biofilm has a higher hydrogen production ability than in the case where it is dispersed in the culture solution. Therefore, hydrogen can be efficiently produced by culturing cells in the state of a biofilm.

(Hydrogen production equipment)
An example of the hydrogen production apparatus of the present invention is shown in FIG.
The culture tank 10 is where the cells are cultured. The culture tank 10 includes a support 11, microbial cells 12, and a culture solution. On the liquid surface of the culture solution, there are inert gas and hydrogen produced by the cells 12. Moreover, the heating means 13 is provided in the circumference | surroundings (for example, side surface and bottom face) of the culture tank 10, and the temperature in the culture tank 10 can be controlled to 60-105 degreeC. Specific examples of the heating means 13 include a silicon rubber heater and a mantle heater.
In FIG. 1, the support 11 is represented by a rectangle, the microbial cell 12 is represented by a circle, and the biofilm is represented by a hexagon.

  The culture tank 10 cultivates Thermococcus kodakaraensis, which is a hyperthermophilic bacterium, and therefore needs to be able to withstand a temperature of about 100 ° C. Moreover, since Thermococcus kodakaraensis is anaerobic, it needs to have a low oxygen permeability. Moreover, since a high concentration salt may be used for the culture solution supplied into the culture tank 10 and hydrogen sulfide may be generated during the cultivation of the hyperthermophilic bacteria, the culture tank 10 is It must be highly resistant to corrosion. Therefore, as the material of the culture tank 10, acid-resistant metals such as aluminum, stainless steel, and hastelloy having the above characteristics are used. Among the stainless steels, austenitic stainless steel is preferable, and SUS316L, SUS304, and the like are particularly preferable because they are inexpensive and easily available.

The material exemplified above is preferably coated or lined with a resin. Thereby, corrosion resistance can further be improved. Examples of this resin include resins consisting only of carbon and hydrogen (for example, polyethylene and polypropylene), but a fluororesin is preferred because of its high heat resistance. Among these fluororesins, polytetrafluoroethylene (PTFE), polychlorotrifluoroethylene (PCTFE), tetrafluoroethylene-perfluoroalkyl vinyl ether copolymer (PFA), tetrafluoroethylene-hexafluoropropylene copolymer ( FEP) and the like are particularly preferable.
In addition, the shape of the culture tank 10 should just be a thing suitable for culture, for example, a cylindrical shape is mentioned.

At the bottom of the culture tank 10, there is provided a culture liquid supply means 20 including a culture liquid storage tank 21 and a culture liquid supply pipe 22 that supplies the culture liquid in the culture liquid storage tank 21 to the culture tank 10. . The culture tank 10 and the culture solution storage tank 21 are connected via a culture solution supply pipe 22. The culture solution is supplied to the culture tank 10 by the culture solution supply means 20.
In the present invention, the “bottom” refers to the end of the culture tank 10 in the direction of gravity, and the “top” described below refers to the end of the culture tank 10 in the direction opposite to the direction of gravity.

Provided on the side surface of the culture tank 10 is a waste liquid recovery means 30 comprising a waste liquid recovery pipe 32 capable of recovering the culture liquid in the culture tank 10 as waste liquid and a waste liquid recovery tank 31 connected to the waste liquid recovery pipe 32. It has been. The culture tank 10 and the waste liquid collection tank 31 are connected via a waste liquid collection pipe 32. By this waste liquid recovery means 30, the waste liquid generated in the culture tank 10 is recovered.
In the present invention, the “waste liquid” refers to a culture liquid in which an amino acid source and a carbon source are consumed by the bacterial cells 12 and contain the excrement of the bacterial cells 12.

A pH electrode 33 is attached to the waste liquid recovery pipe 32, and this pH electrode 33 is connected so as to be able to communicate with the control device 34. Since the microbial cells 12 metabolize the carbon source and excrete acids such as acetic acid, the pH of the culture solution is usually lowered (the degree of acidity is increased). Therefore, by measuring the pH of the waste liquid using the pH electrode 33 and the control device 34, it is possible to evaluate the use state of the culture liquid.
The recovered waste liquid may be discarded as it is. Moreover, after measuring and adjusting pH, you may supply to the culture tank 10 again. By reusing the waste liquid in this way, the utilization efficiency of the culture liquid can be improved.

  At the top of the culture tank 10, a hydrogen gas recovery system comprising a hydrogen gas recovery pipe 42 capable of recovering a gas containing hydrogen in the culture tank 10 and a hydrogen gas recovery tank 41 connected to the hydrogen gas recovery pipe 42. Means 40 are provided. The culture tank 10 and the hydrogen gas recovery tank 41 are connected via a hydrogen gas recovery pipe 42. The hydrogen gas recovery means 40 recovers the gas in the culture tank 10 such as inert gas and hydrogen produced by the bacterial cells 12. Note that a gas chromatograph analyzer may be attached to the hydrogen gas recovery means 41. Thereby, the component of the recovered gas can be measured.

At the bottom of the culture tank 10, an alkaline solution supply means 50 including an alkaline solution storage tank 51 and an alkaline solution supply pipe 52 that supplies the alkaline solution in the alkaline solution storage tank 51 to the culture tank 10 is provided. . The culture tank 10 and the alkaline solution storage tank 51 are connected via an alkaline solution supply pipe 52. The alkaline solution supply means 50 supplies an alkaline solution to the culture tank 10. The alkaline solution is not particularly limited, and examples thereof include an aqueous sodium hydroxide solution.
As described above, since the pH of the culture solution decreases due to the metabolism of the bacterial cells 12, the pH of the culture solution is increased by supplying the culture solution from the culture solution supply unit 20 and discharging the waste solution to the waste solution collection unit 30. It is necessary to maintain bacterial metabolism. If the pH is below the preferred range even when supplying a new culture solution, the alkaline solution is supplied from the alkaline solution supply means 50 to the culture tank 10 to adjust the pH of the culture solution.
In addition, when supplying an alkaline solution into the culture tank 10, the dissolved oxygen in an alkaline solution is substituted by inert gas, such as nitrogen and argon, beforehand. Thereby, it can prevent that culture | cultivation is inhibited by oxygen.

In FIG. 2, the formation process of the biofilm by the support body 11 and the microbial cell 12 in the culture tank 10 of the hydrogen production apparatus of this invention is shown. Also in FIG. 2, as in FIG. 1, the support 11 is represented by a rectangle, the microbial cell 12 is represented by a circle, and the biofilm is represented by a hexagon.
FIG. 2A shows a state in the culture tank 10 in the initial stage of culture. In the culture tank 10, the support body 11 and the microbial cell 12 are disperse | distributing, and recirculate | circulate along the arrow direction of a figure with the culture solution supplied from the bottom part, for example. That is, a fluidized bed is formed. A part of the microbial cell 12 is adsorbed to the support 11.

  FIG. 2B shows a state in the culture tank 10 in the middle stage of the culture. As the culture progresses, a biofilm is formed on the support 11. This biofilm adheres to other biofilms and is deposited in the lower part of the culture tank 10 by its own weight. On the other hand, in the upper part of the culture tank 10, the support body 11 and the microbial cell 12 which are disperse | distributing in the culture solution are recirculating along the arrow direction of a figure. That is, both a fixed bed and a fluidized bed are formed.

FIG. 2C shows a state in the culture tank 10 in the later stage of culture. At this stage, the biofilm is deposited close to the liquid level of the culture solution. That is, a fixed floor is formed. The cells 12 are further cultured and densified in a fixed bed.
Thus, according to the hydrogen production apparatus of the present invention, it is possible to easily achieve the immobilization and densification of the bacterial cells 12. Moreover, hydrogen can be manufactured with a high production rate and high productivity by using the microbial cell 12 fixed at high density.

  Hereinafter, the present invention will be described in more detail by way of examples. The present invention is not limited to the following examples.

(Example 1) Immobilization of bacterial cell 12 using support 11 In Example 1, the bacterial cell 12 was immobilized using the support 11.
(1) Culture of KOD1 strain First, 800 ml of culture solution of medium composition A was filled in a sealed glass container having an internal volume of about 1,000 ml and subjected to high-pressure steam sterilization (121 ° C., 20 minutes). Then, using an anaerobic culture device (model: EAN-140, manufactured by Tabai Espec), add KOD1 strain (8.0 ml of the seed solution) so that the inoculum is 1% by volume under anaerobic atmosphere. Subsequently, the cells were cultured at 85 ° C. for 14 hours in a dryer (program oven, model: MOV-212P, manufactured by Sanyo Electric Co., Ltd.).

  Medium composition A consists of 30.4 g / l of artificial seawater for marine art SF-1 test research (manufactured by Tomita Pharmaceutical Co., Ltd.) (diluted 0.8 times the specified amount), 5 g / l of special dry yeast extract for microbial medium (Nacalai Tesque) Co., Ltd.), special tryptone for microorganism culture medium 5 g / l (manufactured by Nacalai Tesque), sodium pyruvate 5 g / l (manufactured by Nacalai Tesque), and water. Marine art is a mixture of salts containing the same components as seawater, and tryptone is obtained by partially hydrolyzing protein with trypsin.

  The KOD1 strain was continuously cultured by a conventional method using a culture solution in which the concentration of the carbon source (sodium pyruvate) and the amino acid source (tryptone) in the medium composition A was doubled. In comparison, the bacterial cell concentration was improved by 28% and the hydrogen generation rate was improved by 56%. Further, in the medium composition B in which the carbon source in the medium composition A was changed to maltodextrin 5 g / l (trade name: Amicol No. 3-L, manufactured by Nissho Chemical Co., Ltd.), the carbon source (maltodextrin) and amino acid source ( When the KOD1 strain was continuously cultured by a conventional method using a culture solution in which the concentration of tryptone) was doubled, the cell concentration was 27% and the hydrogen generation rate was 52% compared to the culture solution of medium composition B. Improved. In addition, when a culture solution in which the concentration of the carbon source (maltodextrin) and amino acid source (tryptone) is tripled is used, the cell concentration is 40% compared to the culture solution of the medium composition A, and hydrogen is generated. Speed increased by 83%. From these results, it was found that the essential components of Thermococcus kodakaraensis involved in hydrogen production are a carbon source and an amino acid source.

  The inoculum solution is a cell-preserving solution obtained by culturing the KOD1 strain with a culture solution in a 85 ° C. dryer (program oven, model: MOV-212P, manufactured by Sanyo Electric Co., Ltd.) for 10 hours. This culture solution is composed of 30.4 g / l of artificial seawater for marine art SF-1 test and research (manufactured by Tomita Pharmaceutical Co., Ltd.) (diluted 0.8 times the specified amount), and 5 g / l of special dry yeast extract for microbial medium. (Manufactured by Nacalai Tesque Co., Ltd.), special tryptone for microorganism culture medium 5 g / l (manufactured by Nacalai Tesque Co., Ltd.), sulfur powder 5 g / l (manufactured by Wako Pure Chemical Industries Ltd.) 121 ° C., 20 minutes). Sulfur was added to the culture solution after sterilization.

The autoclave sterilization process was performed using an autoclave SX-500 (manufactured by Tommy Seiko Co., Ltd.) under the conditions of 121 ° C., 20 minutes, and 0.1 MPa pressure.
The anaerobic atmosphere in the anaerobic culture apparatus (model: EAN-140, manufactured by Tabai Espec) was prepared using a nitrogen gas base and a 3% hydrogen mixed gas (manufactured by Japan Air Gases Co., Ltd.).

(2) Preparation of culture medium containing support 11 Six bottles filled with 80 ml culture medium of medium composition A were prepared in a sealed glass container having an internal volume of about 300 ml, and subjected to high-pressure steam sterilization (121 ° C., 20 minutes) )did. Thereafter, one type of support E from support A was added to five sealed glass containers in an anaerobic atmosphere using an anaerobic culture apparatus. In FIG. 3, the support body E to the support body E is shown. Note that the support 11 was not added to the remaining one sealed glass container. Six sealed glass containers were placed in an incubator (model: MIR-162, manufactured by Sanyo Electric Co., Ltd.) at 60 ° C. for 2 hours to equilibrate the support 11 in the culture solution.

The support A is a commercially available sponge and the material is polyurethane foam. The support B is a sponge for wastewater treatment (manufactured by Biocarrier Technology Co., Ltd., using fluidized bed carrier ligand carrier, for immobilizing microorganisms such as BOD treatment). The support C is activated carbon (manufactured by Nacalai Tesque Co., Ltd., granular (manufactured by bituminous coal), particle size: 8 to 30 mesh). The support D is an ion exchange resin, Diaion HP20 (manufactured by Mitsubishi Chemical Corporation, effective diameter 0.25 mm or more, particle size distribution 250 μm is 90% or more). The support E is ceramic (manufactured by Kubota Corporation, original product).
In addition, the processing capacity of the support B is 13 g-carrier amount / L-drainage amount, and it is necessary to grasp the volume of the sealed glass container and the adsorption characteristics of the cells 12 to the support 11 over time. The amount of 11 was 1.0 g.

(3) Culture of KOD1 strain using culture medium containing support 11 The culture liquid of (1) cultured for 14 hours in an anaerobic atmosphere using an anaerobic culture apparatus was prepared in (2) 6 120 ml was added to each sample with a graduated cylinder. Thereafter, batch culture was performed at 85 ° C. for 2 hours in a dryer in order to familiarize the culture solution containing the support 11 and the bacterial cells 12.

(4) Measurement of bacterial cell concentration After batch culture in (3), 6 samples were left at room temperature to stop the growth of bacterial cell 12. Each sample was agitated about once every 8 hours, and the culture solution containing the support 11 and the microbial cells 12 was adapted. 5 ml of the supernatant of each sample was sampled periodically in an anaerobic atmosphere using an anaerobic culture apparatus, and the wavelength was 660 nm using a digital colorimeter simple OD monitor (model: mini photo 518R, manufactured by Taitec Corporation). The absorbance was measured. Thereby, the bacterial cell concentration was measured.

The bacterial cell concentration was determined by the following formula.
Cell concentration [g / l] = absorbance value at a wavelength of 660 nm [−] × 0.35 [g / l] (in the case of medium composition A)
By periodically measuring the cell concentration of the supernatant of the sample, changes over time in which the cells 12 were immobilized (adsorbed) on the support 11 were grasped, and the support 11 was evaluated.

  FIG. 4 shows a graph of the cell concentration against the culture time. From FIG. 4, it was found that the concentration of the bacterial cells 12 in the supernatant of the sample having the support 11 decreased after 50 hours from the start of the culture. In particular, when Support A and Support B were used, it was found that the concentration of the bacterial cells 12 in the supernatant was reduced by 50% or more. This result shows that the bacterial cells 12 are immobilized on the support 11 over time.

  The sponge for wastewater treatment of the support B is coated with a microbial adsorbent that has the function of adsorbing microorganisms (activated sludge) on the surface of the material and promoting immobilization, so that it has excellent immobilization ability it is conceivable that.

Example 2 Production of Biofilm and Hydrogen Using Support 11 In Example 2, a biofilm and hydrogen were produced using the support 11.
(1) Preparation of Support 11 As the support 11, a sponge (hereinafter simply referred to as “sponge”) having a heat resistant temperature of about 90 ° C. and a material made of melamine resin was used. The sponge was cut into approximately 5 mm squares to increase the surface area. This facilitated immobilization of the cells 12 and facilitated the formation of a biofilm with the sponge as a nucleus.

(2) Description of the culture apparatus As the culture apparatus, a table type anaerobic thermophilic culturing apparatus manufactured by Taiyo Nippon Sanso Co., Ltd. (culture tank inner product: 1.0 L, with heating means for side and bottom heaters) is used. Using.

(3) Composition of culture solution As the culture solution, one having medium composition B was used.
Medium composition B is 30.4 g / l of artificial seawater for marine art SF-1 test and research (normal amount 0.8-fold dilution), 5 g / l of special dry yeast extract for microbial medium, 5 g / l of special tryptone for microbial medium, Maltodextrin 5 g / l (trade name: Amicol No. 3-L, manufactured by Nissho Chemical Co., Ltd.), 1% adecanol aqueous solution 0.5 ml / l-culture solution (stock solution is Adecanol LG-109, Asahi Denka Kogyo Co., Ltd.) Product), and water. Note that the 1% adecanol aqueous solution is an antifoaming agent, and has an effect of preventing the liquid level control device from malfunctioning when the bacterial cells 12 produce biogas and the culture solution is foamed during continuous culture.

(4) Culture preparation 500 ml of the culture solution shown in the medium composition B was filled in the culture tank of the culture apparatus. This culture tank was autoclaved (121 ° C., 20 minutes), and 0.5 g of the sponge prepared in (1) was added to the culture tank. After that, in order to reduce dissolved oxygen in the sponge and culture medium and to make the inside of the culture tank under an anaerobic atmosphere, an inert gas supply means (digital mass flow controller model attached to the culture apparatus: SEC-E40, manufactured by ESTEC Co., Ltd.) Then, nitrogen was aerated at 100 ml / min into the culture solution. As nitrogen, industrial nitrogen (manufactured by Taiyo Nippon Sanso Corporation) was used.
In order to equilibrate and adapt the sponge with the culture solution, the inside of the culture vessel was stirred at a stirring speed of 50 rpm using a stirring blade attached to the culture apparatus. By attaching a SUS net (material: 18-8 stainless steel) to the stirring blade, physical influence (for example, shear stress) by the stirring blade was prevented, and a biofilm was easily formed using a sponge as a core. In order to sterilize the sponge-friendly environment and sponge, the temperature in the culture tank was controlled at 85 ° C. using the side heater, bottom heater, and temperature sensor (side temperature resistor) attached to the culture apparatus.

(5) Pre-culture operation In pre-culture, 200 ml of culture solution of medium composition B was filled in a sealed glass container having an internal volume of about 300 ml and subjected to high-pressure steam sterilization (121 ° C., 20 minutes). Then, KOD1 strain (2.0 ml of inoculum solution) was added so that the inoculation amount was 1% by volume in an anaerobic atmosphere using an anaerobic culture apparatus, and then 85 ° C. for 11 hours in a dryer. Batch culture was performed.

(6) Batch culture operation 150 ml of the pre-cultured culture solution of (5) was inoculated into a culture tank filled with 0.5 g of the sponge prepared in (4) and 500 ml of culture medium of medium composition B (30% by volume planting). Conduct fungus). Even after inoculation, batch culture was performed for 2 hours under the same conditions as the culture preparation in (4).
During batch culture, about 5 to 20 ml of the culture solution in the culture tank was sampled periodically using the pump attached to the culture apparatus, and the absorbance at a wavelength of 660 nm was measured using a digital colorimeter simple OD monitor. Thereby, the cell density | concentration of the culture solution was measured.

The bacterial cell concentration was determined by the following formula.
Cell concentration [g / l] = absorbance value at a wavelength of 660 nm [−] × 0.53 [g / l] (in the case of medium composition B)
During batch culture, part of the gas that periodically passed through the culture tank is sent to a gas chromatograph analyzer (GC-TCD thermal conductivity detector: GC-8A type, manufactured by Shimadzu Corporation), and its components are It was measured.

(7) Continuous culture operation After performing batch culture for 2 hours, continuous culture was started. Specifically, the culture solution of the medium composition B after high-pressure steam sterilization (121 ° C., 20 minutes) using a master flex digital liquid pump (7524-40 type, manufactured by Yamato Scientific Co., Ltd.) Was continuously fed into. Using a liquid level sensor attached to the culture apparatus and a quantitative liquid feeding pump (RP-1000 type, manufactured by Tokyo Science Instruments Co., Ltd.), the culture liquid in the culture tank was controlled to a liquid level of 500 ml. In addition, the cell density | concentration of the culture solution in a culture tank after completion | finish of batch culture was 0.1 g / l or more.

  Simultaneously with the start of continuous culture, acid (using 0.2 to 0.4N HCl aqueous solution) is supplied using a pH composite electrode (model: DPZ-220, manufactured by Maruhishi Bio-Engine Co., Ltd.), alkaline (0.2-0.4 mol / l NaOH aqueous solution was used) was supplied and controlled to pH 6.4. The aqueous HCl solution and aqueous NaOH solution were degassed with nitrogen gas. As nitrogen, industrial nitrogen (manufactured by Taiyo Nippon Sanso Corporation) was used. The supply flow rate of nitrogen gas was about 50 ml / min. During the continuous culture, the bacterial cell concentration and gas analysis were periodically performed in the same manner as the batch culture operation of (6).

The culture solution of medium composition B was also aerated with nitrogen gas using a digital mass flow controller (model CMQ0002, manufactured by Yamatake Corporation). The supply flow rate of nitrogen gas was 100 ml / min. Moreover, HCl aqueous solution, NaOH aqueous solution, and culture solution were mixed with aeration.
As continuous culture conditions, a temperature of 85 ° C., a stirring speed of 50 rpm, pH 6.4, and a nitrogen gas flow rate of 200 ml / min for supplying the culture tank were selected. Continuous culture was performed under these culture conditions. Since the hydrogen produced by the cells 12 inhibits the growth of the cells 12, the nitrogen gas supply flow rate was doubled compared to that during batch culture.

  The cell concentration became stable after about 55 hours from the start of continuous culture. Therefore, at this time, the composition of the culture solution to be supplied was changed from the medium composition B to the medium composition C in order to increase the production amount of biofilm and hydrogen.

  The medium composition C is the same as the medium composition B except that it has 10 g / l each of a special tryptone for microorganism medium and maltodextrin. The medium composition C was twice the concentration of maltodextrin and tryptone compared to the medium composition B. The culture medium of medium composition C was also subjected to high-pressure steam sterilization (121 ° C., 20 minutes) and then supplied to the culture tank using a digital mass flow controller while mixing the degassed / cultured liquid with nitrogen gas. The supply flow rate of nitrogen gas was about 100 ml / min.

The dilution rate represents the average number of times the culture medium in the culture tank is replaced with a fresh medium (nutrient source) per unit time. The greater the dilution rate, the greater the dilution of the bacterial cells 12 in the culture tank. The dilution rate D was determined by the following formula.
Dilution rate D [hr ⁻¹ ] = fresh medium (nutrient source) supply rate [L / hr] / culture liquid amount [L]
Increasing the dilution rate not only activates cell division of the cells and improves the amount of hydrogen production, but also increases the amount of polysaccharide produced when the carbon source is metabolized. Therefore, a biofilm is easily formed. Therefore, the dilution rate and the biofilm size are in a proportional relationship. Therefore, by increasing the dilution rate of the medium composition C, the growth rate of the bacterial cells 12 was also increased, and the biofilm production amount and the hydrogen production amount were increased. Specifically, the dilution rate was first set to 0.3, and then increased in order to 0.6 and 1.2 [hr ⁻¹ ].

(8) Observation of biofilm Using a field emission scanning electron microscope (S-4700, manufactured by Hitachi, Ltd.), the produced biofilm was observed, and the cells 12 were immobilized in the biofilm. Checked whether or not.

(9) Result 1 (Manufacture of biofilm)
As a result of continuous culture (operations (1) to (7) in Example 2) using a sponge, a biofilm could be produced. 5 and 6 show photographs of the obtained biofilm. The total culture time from the inoculation to the end of continuous culture was 162 hours.
As a comparative example, a conventional continuous culture without using a sponge (the operation of (2) to (7) without using the sponge of Example 2) was performed, and a biofilm having a shape as shown in FIGS. 5 and 6 was produced. could not.
Therefore, it became clear that a biofilm can be produced specifically by using a sponge.

(10) Result 2 (observation of biofilm, confirmation of immobilization of bacterial cells 12)
FIG. 7 shows a field emission scanning electron microscope image of the sponge used as the support 11. FIG. 8 shows a field emission scanning electron microscope image of the biofilm (2) of FIG. 5 (a sample in which the biofilm is not formed on the sponge by visual observation). FIG. 9 shows a field emission scanning electron microscope image of the biofilm (3) in FIG. 5 (a sample in which a biofilm is formed using a sponge as a nucleus when observed with the naked eye).

  From the comparison between FIG. 7 and FIG. 8, it was confirmed that the bacterial cells 12 were immobilized on the sponge. Moreover, it was confirmed from the comparison of FIG. 7 to FIG. 9 that the biofilm grows with the sponge as a nucleus. It became clear that the microbial cells 12 were fixed and densified in proportion to the culture time, and the microbial cell concentration increased.

FIG. 10 shows a graph of the bacterial cell concentration against the dilution rate.
From FIG. 10, when the conventional continuous culture which does not use sponge was performed, the microbial cell density | concentration of the culture solution in a culture tank fell by the influence of the dilution by a nutrient source supply rate raise. On the other hand, when continuous culture using a sponge was performed, the bacterial cell concentration in the culture solution in the culture tank did not change, and was higher than that of conventional continuous culture without using a sponge. In addition, although the microbial cell density | concentration at the time of using sponge is apparently stable, since the microbial cell 12 is fix | immobilized in the biofilm, it is estimated that the microbial cell density | concentration in a culture tank is substantially high. The

(11) Result 3 (production of hydrogen)
FIG. 11 shows a graph of the hydrogen generation rate against the dilution rate.
From Figure 11, when subjected to continuous culture using a sponge, when the dilution rate D is 1.1 ^{[hr -1],} that hydrogen generation rate is _0.5L-H 2 / hr · l- culture Became clear. In addition, the hydrogen generation rate per unit time and unit volume when performing continuous culture using a sponge is D = 1.1 [hr ^{− as} compared with the case of performing conventional continuous culture without using a sponge. ¹ ] was found to increase by 57%.

  From this result, it was shown that if the sponge amount of the support 11 is increased, the biofilm production amount and the bacterial cell concentration are increased, and the hydrogen production amount (hydrogen generation rate) is also increased. Moreover, by increasing the nutrient source concentration or / and dilution rate in the culture solution, the growth rate of the bacterial cells 12 is increased, the production amount of the biofilm and the bacterial cell concentration are increased, and the hydrogen production amount (hydrogen generation rate) is also increased. It was shown to be.

  According to the present invention, the production rate and productivity can be improved in the production of hydrogen using hydrogen-producing anaerobic hyperthermophilic bacteria such as Thermococcus kodakaraensis. This hydrogen is useful as an energy source used in power generators such as fuel cells. Moreover, as a raw material of the culture solution, factory waste water or domestic waste water rich in amino acid sources and carbon sources can be used. Thus, the present invention is also useful in the waste treatment industry.

It is the schematic which shows an example of the hydrogen production apparatus concerning embodiment of this invention. It is a schematic diagram which shows the formation process of the biofilm by the support body 11 and the microbial cell 12 in the culture tank 10 of the hydrogen production apparatus of this invention. 2 is a photograph of supports A, B, C, D, and E used in Example 1. FIG. 2 is a graph of bacterial cell concentration versus culture time in Example 1. FIG. 2 is a photograph of a biofilm obtained in Example 2. 2 is an enlarged photograph of a biofilm obtained in Example 2. 3 is a field emission scanning electron microscope image of a sponge in Example 2. FIG. 6 is a field emission scanning electron microscope image of the biofilm of FIG. 5 (2) in Example 2. FIG. 6 is a field emission scanning electron microscope image of the biofilm of FIG. 5 (3) in Example 2. FIG. 6 is a graph of bacterial cell concentration versus dilution rate in Example 2. 6 is a graph of hydrogen generation rate versus dilution rate in Example 2.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 10 ... Culture tank, 11 ... Support body, 12 ... Hydrogen generating anaerobic hyperthermophilic bacterium (bacteria), 20 ... Culture solution supply means, 30 ... Waste liquid collection means, ..Hydrogen gas recovery means, 50 ... alkali solution supply means

Claims (8)

  A method for producing hydrogen, wherein a hydrogen-producing anaerobic hyperthermophilic bacterium is cultured using a support and a culture solution containing an amino acid source and a carbon source.   The method for producing hydrogen according to claim 1, wherein the support is porous.   The support is natural sponge, synthetic sponge, activated carbon, ceramic, ion exchange resin, zeolite, foamed stone, molten slag, cellulose acetate fiber, and glass beads, carbon, silica gel, sand, alumina, molecular sieve, and titanium oxidation. The method for producing hydrogen according to claim 1 or 2, wherein the method is one or more selected from the group consisting of products.   The method for producing hydrogen according to any one of claims 1 to 3, wherein the carbon source is one or more selected from the group consisting of pyruvic acid, pyruvate, and starch-based polysaccharides.   The method for producing hydrogen according to any one of claims 1 to 4, wherein the hydrogen-producing anaerobic hyperthermophilic bacterium is Thermococcus kodakaraensis.   The method for producing hydrogen according to claim 5, wherein the Thermococcus kodakaraensis is Thermococcus kodakaraensis KOD1. A culture tank filled with a support and hydrogen-producing anaerobic hyperthermophilic bacteria;
A culture solution supply means for supplying a culture solution containing an amino acid source and a carbon source to the culture tank;
Waste liquid recovery means for recovering waste liquid from the culture tank;
Hydrogen gas recovery means for recovering hydrogen gas generated in the culture tank;
A hydrogen production apparatus comprising:   The hydrogen production apparatus according to claim 7, further comprising alkaline solution supply means for supplying an alkaline solution to the culture tank.

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