JP4476285B2 - Microbial culture apparatus, hydrogen production apparatus and fuel cell system using the same - Google Patents

Description

  The present invention relates to a microorganism culturing apparatus, a hydrogen production apparatus using microorganisms cultured by the microorganism, and a fuel cell system using the same.

  Unlike fossil raw materials, hydrogen is attracting attention as the ultimate clean energy source that does not generate substances that are concerned due to environmental problems such as carbon dioxide and sulfur oxides even when burned. The amount of heat per unit mass of hydrogen is more than three times that of petroleum, and if it is supplied to a fuel cell, it can be converted into electric energy and heat energy with high efficiency.

  As a method for producing hydrogen, techniques such as a pyrolysis steam reforming method of natural gas or naphtha have been conventionally proposed as a chemical production method. This method requires high temperature and high pressure reaction conditions. Further, since the synthesis gas produced by this method contains CO (carbon monoxide), when used as a fuel for fuel cells, it is highly difficult to solve technical problems in order to prevent deterioration of the fuel cell electrode catalyst. It is necessary to perform CO removal.

On the other hand, in the method for producing biological hydrogen by microorganisms, the reaction conditions are normal temperature and normal pressure, and the generated gas does not contain CO, so that removal thereof is unnecessary.
From such a viewpoint, biological hydrogen production by microorganisms is more preferable as a fuel (hydrogen) supply method for fuel cells, and has attracted attention.

  Biological hydrogen production methods are roughly classified into methods using photosynthetic microorganisms and methods using non-photosynthetic microorganisms (mainly anaerobic microorganisms). The former method uses light energy for hydrogen generation. However, since the light energy utilization efficiency of photosynthetic microorganisms is low, a large condensing area is required for hydrogen generation. In addition, there are many problems that need to be solved, such as the price issue of the hydrogen generator and the difficulty of maintenance, and it has not reached a practical level.

  Regarding the latter hydrogen production method using anaerobic microorganisms, Japanese Patent Application Laid-Open No. 2003-135088 (Patent Document 1) mainly separates hydrogen from biogas generated in a hydrogen fermenter, and mainly uses the remaining carbon dioxide. The residual gas as a component is circulated into the hydrogen fermenter and blown into the fermentation broth, and the hydrogen concentration is reduced by pressurizing the fermenter to activate the action of hydrogen-producing bacteria. A method for increasing the hydrogen yield is disclosed.

  In addition, regarding a system with a fuel cell using hydrogen produced using microorganisms, Japanese Patent Laid-Open No. 2002-280045 (Patent Document 2) supplies hydrogen gas generated by microorganisms to a fuel cell and consumes hydrogen. A fuel cell built-in bioreactor is shown in which hydrogen gas is sucked into the fuel cell from the gas phase portion of the reactor by lowering the hydrogen partial pressure at the hydrogen inlet according to

  Furthermore, as a post-treatment method after hydrogen fermentation, in Japanese Patent Application Laid-Open No. 2002-355022 (Patent Document 3), in the first reaction vessel into which a Clostridium microorganism is introduced, the Clostridium microorganism is an organic material. Hydrogen is produced by decomposition, the remaining organic material after hydrogen generation is introduced into the second reaction vessel, and the second microorganism is introduced after the addition of the organic material, thereby converting the organic material into water and carbon dioxide. Can be broken down into Thus, a method is shown in which almost no emissions (dust) are generated by the decomposition of the organic material in the second reaction vessel.

In addition, regarding a high-density culture method for microorganisms, Japanese Patent Application Laid-Open No. 11-99397 (Patent Document 4) uses a microbial cell (aerobic bacterium) to treat treated water from an aeration tank into a membrane separation device. Introducing a culture method for culturing at a high density by removing moisture out of the system by introducing water through the membrane and returning the non-permeate not permeating the membrane to the aeration tank.
JP 2003-135088 A JP 2002-280045 A Japanese Patent Laid-Open No. 2002-355022 Japanese Patent Laid-Open No. 11-99397

In conventional hydrogen production equipment using microorganisms, the hydrogen production capacity of microorganisms decreases during hydrogen production, so the microorganisms in the hydrogen production equipment are removed for the purpose of recovering the hydrogen production capacity. It was necessary to exchange and replenish. However, when microorganisms are exchanged / replenished, hydrogen production is temporarily stopped, and hydrogen cannot be used. For example, if the hydrogen is used for power generation in a fuel cell, electricity cannot be used, which is very inconvenient. Moreover, since the microorganisms to be replaced / replenished have a short storage period and the hydrogen generation ability decreases with the passage of time, it is necessary to frequently replace / replenish them, which is very labor intensive.
When microorganisms are stored, it is necessary to store them under low temperature conditions, a special storage is necessary from the viewpoint of safety, and it is necessary to periodically purchase microorganisms. In addition, there are problems such as complicated processing of containers storing microorganisms.
Thus, in response to the problem, continuously culturing a large amount of microorganisms having hydrogen generating ability, continuously inducing the hydrogen generating ability of the microorganisms, and continuously supplying the microorganisms to the hydrogen production apparatus No device has been found that can.

  In addition, as a conventional method of producing hydrogen using microorganisms, the method of producing hydrogen by fermenting organic substances increases the amount of hydrogen generated and the hydrogen yield by lowering the hydrogen concentration in the reaction vessel. Has been shown to let So far, in hydrogen production using microorganisms, methods for increasing the amount of hydrogen generation and methods for improving the hydrogen yield have been studied. However, in a hydrogen production apparatus that uses microorganisms, it is difficult to control the hydrogen generation amount because the amount of hydrogen generation obtained is small, so no hydrogen production apparatus that can control the hydrogen generation amount has been found. It was.

  Furthermore, in a system comprising a hydrogen production apparatus using a microorganism and a fuel cell, Japanese Patent Application Laid-Open No. 2002-280045 discloses a hydrogen production apparatus using a microorganism that maintains the hydrogen partial pressure at a low level by utilizing the consumption of hydrogen by power generation of the fuel cell. And a system including a fuel cell is shown. However, a fuel cell system including a hydrogen production device that can control the amount of power generation by controlling the amount of hydrogen produced, that is, a fuel that can be used at a practical level by a hydrogen production method using microorganisms. No battery system has been found so far.

  Moreover, in the conventional hydrogen production method using microorganisms, there was a problem of processing of fermentation residues after hydrogen generation. In Japanese Patent Laid-Open No. 2002-355022, two reaction vessels are used to generate hydrogen in the first reaction vessel, and the remaining organic material after the hydrogen generation is charged into the second reaction vessel, and further the second microorganism Shows the method of decomposing water and carbon dioxide, and generating no emissions. However, there is a problem that the process is complicated and control is difficult, for example, it is necessary to put the remaining organic material after hydrogen generation into the second reaction vessel.

Various metabolic pathways for hydrogen generation in anaerobic microorganisms are known. Examples of such pathways include a pathway for generating hydrogen in the degradation pathway of glucose to pyruvate, a pathway for generating hydrogen in the process of generating acetic acid from pyruvate via acetyl CoA, and a formate derived from pyruvate. A route for generating hydrogen more directly is included.
A typical reaction formula for hydrogen generation is shown below.

(A) Degradation pathway of glucose to pyruvate
C ₆ H ₁₂ O ₆ (glucose) → 2CH ₃ COCOOH (pyruvic acid) + 2H ₂ (hydrogen)
(B) Pathway in which acetic acid is generated by pyruvic acid via acetyl-CoA
CH ₃ COCOOH + H ₂ O → CH ₃ COOH (acetic acid) + CO ₂ (carbon dioxide) + H ₂
(C) Path through which hydrogen is generated from formic acid
HCOOH (formic acid) → H ₂ + CO ₂

The route (a) is a method via reduced nicotinamide adenine dinucleotide (NADH) and ferredoxin (Fd). Since this route is disadvantageous thermodynamically, it is necessary to reduce the hydrogen partial pressure in the reaction system.
The route (b) is a method via reduced ferredoxin and hydrogenase. In this pathway, acetic acid is produced along with hydrogen. In order to perform continuous production of hydrogen, there is a problem that unnecessary acetic acid is by-produced in the reaction solution.
The route (c) is a method via formate dehydrogenase and hydrogenase. Since the product is only a gas at room temperature, hydrogen is easily separated from the reaction solution, and continuous hydrogen production and hydrogen production of continuous hydrogen are possible.

The present inventors can control a hydrogen production apparatus capable of continuous hydrogen production and a hydrogen production amount by mainly utilizing a metabolic pathway in which hydrogen is produced from formic acid and formate in microbial cells. It has been found that a hydrogen production apparatus can be provided.
As a hydrogen production apparatus capable of continuous hydrogen production, by culturing microorganisms continuously under aerobic conditions, allowing the microorganisms to express hydrogen generation ability under anaerobic conditions, and supplying the microorganisms to the hydrogen generation apparatus, Enables continuous hydrogen generation in hydrogen generators. A microorganism culture section communicating with a gas source containing oxygen and a nutrient source supply section; an organic substance supply source for supplying an organic substance that causes the microorganism to express hydrogen generation ability; an anaerobic atmosphere imparting means; And a microorganism producing apparatus comprising a microorganism-producing apparatus having an ability to produce hydrogen under anaerobic conditions, and a hydrogen producing apparatus in which the apparatus is connected to the hydrogen producing apparatus.

  Furthermore, by controlling the supply amount of the organic substrate and controlling at least one hydrogen generation condition selected from formic acid concentration conditions, temperature conditions, pH conditions, microbial concentration conditions, and stirring conditions, hydrogen production We have found a hydrogen production system that can control the amount. In addition, the present inventors have found a fuel cell system using the above hydrogen production apparatus capable of controlling the power generation amount of the fuel cell at the same time. In addition, according to the present invention, it is possible to provide a hydrogen production apparatus that hardly generates a residue, and it is possible to construct a fuel cell system capable of continuously producing hydrogen as a fuel cell system and controlling the power generation amount.

  The present invention relates to a biological hydrogen production apparatus mainly using a metabolic pathway in which hydrogen is generated from formic acid, and a fuel cell system using the same. When the above problems are solved and hydrogen is produced by microorganisms, it is possible to control the hydrogen production amount by controlling the supply amount of an organic substrate that is a raw material for hydrogen production. Furthermore, it is possible to control the amount of hydrogen production by controlling with at least one hydrogen production condition selected from formic acid concentration conditions, temperature conditions, pH conditions, microorganism concentration conditions, and stirring conditions. In addition, in a hydrogen production method using microorganisms, a fuel cell system capable of controlling the amount of power generation by controlling the amount of hydrogen production, that is, a fuel cell system capable of immediately taking out required power is provided. .

  Further, the present invention has found that it is possible to provide a hydrogen production apparatus that hardly generates a residue by selecting an organic substrate. In particular, when formic acid and formate are used as the organic substrate, particularly when formic acid is used, it can be decomposed into hydrogen and carbon dioxide. In that case, since gas is mainly generated, separation from the reaction solution in the reaction tank is easy. Therefore, it is an object of the present invention to provide a hydrogen production apparatus capable of easily and continuously producing hydrogen in one reaction tank without requiring complicated processes and controls.

That is, the present invention provides:
(1) A microorganism culture part communicating with a gas source containing oxygen and a nutrient source supply part, an organic substance supply source for supplying an organic substance that communicates with the culture part and causes the microorganisms to express hydrogen generation ability, and anaerobic atmosphere provision An apparatus for culturing a microorganism, comprising a unit for expressing hydrogen generation capacity under anaerobic conditions of the microorganism having means;
(2) The culture apparatus according to (1) above, wherein a separation unit between the cultured microorganism and the culture solution is provided between the culture unit and the hydrogen production ability expression unit;
(3) The culture apparatus according to (1) or (2) above, wherein the organic substance that causes the microorganism to express hydrogen generation ability is at least one selected from glucose, formic acid and formate;
(4) The culture apparatus according to any one of (1) to (3) above, wherein the microorganism has a formate dehydrogenase gene and a hydrogenase gene;

(5) The culture apparatus according to any one of (1) to (4) above is connected to a hydrogen generation reaction section, and the hydrogen generation reaction section supplies a raw material supply section for supplying an organic substrate. Hydrogen production equipment in communication with
(6) The hydrogen production apparatus according to (5), wherein the production amount of hydrogen is controlled by the supply amount of the organic substrate from the raw material supply unit to the hydrogen generation reaction unit;
(7) The hydrogen production amount is controlled by at least one hydrogen production condition selected from organic substrate concentration conditions, temperature conditions, pH conditions, microorganism concentration conditions and stirring conditions in the hydrogen production reaction section (5) A hydrogen production apparatus according to claim 1;

(8) The hydrogen production apparatus according to (5) above, wherein the organic substrate is supplied to the hydrogen generation reaction section via a spraying apparatus;
(9) Any one of the above (5) to (8), wherein a separation unit for the microorganism and the culture solution that expresses the hydrogen generation capability is provided between the hydrogen generation capability expression unit and the hydrogen generation reaction unit. Hydrogen production apparatus according to
(10) The above (5) to (9), wherein the hydrogen generation reaction unit includes a hydrogen generation amount detection unit, and is configured to be able to supply microorganisms from the culture apparatus based on the detection value of the hydrogen generation amount detection unit. The hydrogen production apparatus according to any one of

(11) The hydrogen production apparatus according to any one of (5) to (10) above, wherein the organic substrate is formic acid or formate;
(12) The hydrogen production apparatus according to any one of (5) to (11) above, wherein the hydrogen generation reaction section includes a condenser for condensing water vapor contained in the discharged gas;
(13) A system comprising the hydrogen production apparatus according to any one of (5) to (12) above and a fuel cell using hydrogen generated from the hydrogen production apparatus as a fuel gas;
(14) The system according to (13), wherein the power generation amount in the fuel cell is controlled by the supply amount of the organic substrate from the raw material supply unit to the hydrogen generation reaction unit;
(15) The power generation amount in the fuel cell is controlled by any one hydrogen generation condition selected from an organic substrate concentration condition, a temperature condition, a pH condition, a microorganism concentration condition and a stirring condition in the hydrogen generation reaction section (13).

  In the present specification, “part” in terms of “nutrient source supply part”, “microbe culture part”, “hydrogen generation ability expression part”, “hydrogen generation reaction part”, etc. means a partitioned space. It may be a tank, a tank, a container or the like. Therefore, hereinafter, for example, “microbe culture section” may be referred to as “microbe culture tank”. These divided spaces may be one or plural.

According to the culture apparatus of the present invention, microorganisms can be continuously cultured, and the microorganisms can continuously develop (reform) hydrogen production ability. In addition, by adding the microorganism culturing apparatus to the hydrogen production apparatus, it is possible to continuously supply microorganisms that have developed hydrogen generating ability to the hydrogen production apparatus, and to provide an apparatus capable of continuous hydrogen production. .
Furthermore, by using the reactor of the present invention in which a microorganism having a formate dehydrogenase gene and a hydrogenase gene is present, the supply amount of the organic substrate and the hydrogen production conditions (organic substrate concentration, temperature, pH, microorganism concentration) And agitation) can be provided to provide a hydrogen production apparatus capable of controlling the amount of hydrogen production. Furthermore, a hydrogen production apparatus capable of controlling the power generation amount of the fuel cell by controlling the supply amount of the organic substrate and the hydrogen generation conditions (organic substrate concentration, temperature, pH, microbial concentration and stirring). A fuel cell system can be provided.
Moreover, the hydrogen production apparatus which can produce hydrogen continuously, and a fuel cell system using the same can be provided.

FIG. 1 is a schematic diagram of a preferred microorganism culture apparatus of the present invention. FIG. 2 is a schematic diagram of a preferred hydrogen generator of the present invention. FIG. 3 is a schematic configuration diagram of a fuel cell system using the hydrogen production apparatus of the present invention. FIG. 4 is a schematic configuration diagram of a fuel cell system using the hydrogen production apparatus used in Examples 2 to 15. FIG. 5 is a graph showing the relationship between the supply rate of the organic substrate using the hydrogen production apparatus of the present invention, the hydrogen production amount, and the power generation amount in the fuel cell system. FIG. 6 is a graph showing the relationship between the concentration of an organic substrate using the hydrogen production apparatus of the present invention, the hydrogen production amount, and the power generation amount in the fuel cell system. FIG. 7 is a graph showing the relationship between the temperature in the reaction solution using the hydrogen production apparatus of the present invention, the hydrogen production amount, and the power generation amount in the fuel cell system. FIG. 8 is a diagram showing the relationship between the pH in the reaction solution using the hydrogen production apparatus of the present invention, the hydrogen production amount, and the power generation amount in the fuel cell system. FIG. 9 is a graph showing the relationship between the microorganism concentration in the reaction solution using the hydrogen production apparatus of the present invention, the hydrogen production amount, and the power generation amount in the fuel cell system. FIG. 10 is a diagram showing the relationship between the rotational speed of a stirrer using the hydrogen production apparatus of the present invention, the hydrogen production amount, and the power generation amount in the fuel cell system.

Explanation of symbols

1: Fuel tank 2: Reaction tank 3: Medium tank 4: Waste liquid tank 5: Hydrogen concentration tank (fixed tank)
6: Fuel cell section 7: Inverter 8: Stirrer 9: Combustion catalyst 10: Clean filter 11: Valve 12: Flow meter 13: Pump 14: Power 15: Medium supply pump 16: Waste liquid extraction port 17: Medium input port 18: Waste liquid extraction pump 19: Air intake port 20: Blower pump 21: Concentrator replacement port (valve)
22: Adsorbent 23: Microbial culture medium tank 24: Culture vessel (culture tank)
25: Sterilization air supply device 26: Reform container (hydrogen generating capacity expression tank)
27: Organic substance supply device 28: Microorganism storage tank 29: Microorganism washing tank 30: Waste liquid valve (vent)
31: Microorganism supply port 33, 35, 37, 40, 42, 44, 45: Pump 32, 34, 36, 39, 41, 43: Flow meter 38: Washing medium supply port 51: Reactor (hydrogen generation reaction part)
52: Tank containing organic substrate 53: Thermostatic bath 54: Fuel cell 55: Condenser 56: Gas separation device 57: Device for spraying organic substrate 58: Tank containing medium components 59: Motor 60: Stirring device 61: Organic substrate supply pump 62: Medium component supply pump 63: Organic substrate supply port 64: Medium component supply port 65: Reaction solution discharge pump 66: Microorganism supply port 67: Various sensors

  The culture apparatus of the present invention includes a microorganism culture tank communicating with a nutrient source supply tank for storing and supplying a gas source containing oxygen and a nutrient source for culturing microorganisms in the culture tank, and a culture solution in which the microorganism is cultured. A microorganism separation unit for separating microorganisms, a reformer tank that has an anaerobic atmosphere imparting means for making the reformer tank anaerobic, and a microorganism that collects and stores microorganisms from the reformer tank It is an apparatus comprising a storage tank.

  The hydrogen generation apparatus of the present invention includes a hydrogen generation reaction tank having a hydrogen discharge port, a tank for storing and supplying a solution containing an organic substrate in the hydrogen generation reaction tank, It is an apparatus comprising a waste liquid tank that removes and stores microorganisms having reduced hydrogen generation ability and metabolic components of the microorganisms, and a fuel cell unit that uses hydrogen generated in a hydrogen generation reaction tank.

The microorganism culture apparatus and hydrogen generator of the present invention will be described below with reference to the drawings.
A preferred embodiment of the culture apparatus of the present invention is shown in FIG.
The microorganism used in the present invention may be any microorganism having hydrogen-producing ability, and is mainly composed of a formate dehydrogenase gene (F. Zinoni et al., Proc. Natl. Acid. Sci. USA, Vol. 83, pp 4650- 4654, July 1986 Biochemistry) and a hydrogenase gene (R. Boehm et al., Molecular Microbiology (1990), 4 (2), 231-243), mainly anaerobic microorganisms.

  Examples of specific anaerobic microorganisms used in the present invention include microorganisms belonging to the genus Escherichia-such as Escherichia coli ATCC 9637, ATCC 11775, ATCC 4157, etc., microorganisms belonging to the genus Klebsiella-such as Klebsiella pneumoniae. (Klebsiella pneumoniae ATCC 13883, ATCC 8044, etc.), Enterobacter microorganisms such as Enterobacter aerogenes ATCC 13048, ATCC 29007 etc. and Clostridium microorganisms such as Clostridium bayerii AT Etc.

  Since the division growth of these anaerobic microorganisms under anaerobic conditions is extremely slow compared to that under aerobic conditions, the microorganisms are preferably grown under aerobic conditions. In this sense, among anaerobic microorganisms, facultative anaerobic microorganisms are preferably used rather than obligate anaerobic microorganisms. Among the above microorganisms, Escherichia coli, Enterobacter aerogenes, and the like can be preferably used.

  In the culturing apparatus of the present invention, the microorganism is cultured under anaerobic conditions to divide and proliferate, and then cultured under anaerobic conditions to produce an enzyme protein involved in the hydrogen production pathway of the microorganism. And thus have the ability to generate hydrogen. The expression of the function of the microorganism in this way is expressed herein as “reforming” (in the present invention).

  The culture unit for microorganisms in the culture apparatus of the present invention communicates with a nutrient source supply unit that stores a nutrient source for growing microorganisms and supplies the nutrient source to the culture unit. In FIG. 1, a microorganism culture tank 24 as a microorganism culture part communicates with a microorganism culture medium tank 23 as a nutrient source supply part.

  As a nutrient source for the growth of microorganisms used in the present invention (hereinafter also referred to as a microorganism growth medium), a normal nutrient medium containing a carbon source, a nitrogen source, a mineral source, and the like can be used. Examples of the carbon source include glucose, fructose, molasses, etc., and examples of the nitrogen source include inorganic nitrogen sources such as ammonia, ammonium salts, and nitrates, and organic nitrogen sources such as urea, amino acids, and proteins. Each can be used alone or in combination. Both inorganic and organic forms can be used in the same manner. Further, as the mineral source, for example, potassium monohydrogen phosphate, magnesium sulfate or the like mainly containing K, P, Mg, S and the like can be used. In addition, nutrients such as various vitamins such as peptone, meat extract, yeast extract, corn steep liquor, casamino acid, biotin, thiamine and the like can be added as necessary. When LB culture medium is used as a culture medium for microorganisms, specifically, a solution prepared by adding water to 10 g of tryptone, 5 g of yeast extract and 10 g of sodium chloride to make 1 liter is supplied to the culture medium tank 23 for microorganism culture. Can do.

The nutrient source supply unit preferably has sterilization means for sterilizing the inside. As the sterilization means, a device capable of controlling the inside of a heater, a heat exchanger or the like to a high temperature can be used.
Thereby, when newly supplying a culture medium to a tank, it can sterilize for about 20 minutes at 120 degreeC by heat processing. However, when a sterilized medium is used in advance, this process may not be performed. It is preferable that a heater, a heat exchanger, or the like is provided on the surface of the microorganism culture medium tank 23.

  It is preferable that the nutrient source supply unit and the microorganism culture unit communicate with each other using at least one connection means selected from a pipe, a container, a pump, a sensor, and a valve. In addition, the culture unit is provided with a mechanism for detecting the amount of liquid in the culture unit, and it is preferable that the nutrient source can be supplied from the nutrient source supply unit based on the detected value.

  1, the microorganism culture medium tank 23 and the culture tank 24 are connected via a flow meter 32 and a pump 33. The pump 33 is connected to the culture tank 24 while measuring the microorganism culture medium using the flow meter 32. Can be used to send.

  Cultivation performed in the microorganism culture section can be generally performed at a temperature of about 20 ° C. to about 40 ° C., preferably about 25 ° C. to about 40 ° C. under aerobic conditions such as aeration and shaking. The pH during the culture is in the range of 5 to 10, preferably 6 to 8. The pH during the culture can be adjusted by adding an acid or an alkali. The carbon source concentration at the start of the culture is 0.1 to 20% (w / v), preferably 1 to 5% (w / v). The culture period is usually from half a day to 5 days.

  The microorganism culture part is in communication with a gas source containing oxygen. In general, oxygen or air is used as the gas containing oxygen. As a gas supply method containing oxygen, a gas supply method used for normal aerobic culture, such as an air pump, can be used. The gas source containing oxygen has a means for sterilizing the gas and can supply the sterilized gas to the culture unit. The amount of sterilized air or oxygen supplied to the microorganism culture part can be appropriately selected according to the microorganism used, but 0.001 to 1 liter oxygen / 1 liter solution · min is preferable. More preferably, it is 0.01 to 0.5 liter oxygen / 1 liter solution · min. In FIG. 1, sterilizing air or oxygen is supplied to the microorganism culture tank from the sterilizing air supply device 25 under the control of the flow meter 34, using a pump 35 at a speed of 0.001 to 1 liter oxygen / 1 liter solution / min. Can send.

  The microorganism culture part communicates with a gas source containing oxygen, and can be cultured under aerobic conditions, but can also be cultured under anaerobic conditions. In this case, no gas is supplied from the gas source to the culture unit.

The microorganism culture part preferably has an introduction port through which the above microorganisms can be introduced. It is preferable that the cells introduced from the introduction port of the microorganisms are introduced so that the cell optical density OD ₆₁₀ = 0.01 to 10 in the culture part of the microorganisms. More preferably, the bacterial cell optical density OD ₆₁₀ = 0.05-5. In FIG. 1, microorganisms are input to the culture tank 24 from a microorganism input port (not shown).

  In the culture apparatus of the present invention, since continuous culture is possible, the microorganisms need only be input at the start of continuous culture. Alternatively, additional microorganisms can be added after the start of continuous culture. Further, when the hydrogen generation ability of microorganisms is reduced due to troubles on the apparatus, the same kind of microorganisms can be introduced from the inlet.

  The microorganisms cultured in the microorganism culture unit are sent to a separation unit that can separate the cultured microorganisms from the culture solution. As a method for separating the microorganism and the culture solution, membrane separation, centrifugation, or the like can be used, and membrane separation that is easy to operate is preferably used. In the separation unit, it is possible to perform an operation of removing a substance that is contained in the culture solution and becomes an inhibition factor when the hydrogen production ability is expressed. Such an operation includes washing the separated microorganisms using an anaerobic unused medium, sterilized water, or the like.

  In FIG. 1, the culture solution sent to the microorganism washing tank 29 as the separation unit is less than 1 μm smaller than the size of the microorganism from the vent 30 in order to remove substances that become an obstacle when inducing hydrogen generation ability. A part or all of the solution other than the microorganisms is separated by the filter of the eye. After the unused medium is supplied from the cleaning medium supply port 38 to the microorganism cleaning tank 29, the mixture of the microorganism and the medium is sent to the reforming container (hydrogen generating capacity expression tank) 26 by the pump 40 and the flow meter 39.

In the culturing apparatus of the present invention, a hydrogen generating ability expression unit (hereinafter also referred to as a reforming unit) for expressing a hydrogen generating ability in a microorganism after culturing includes a system for maintaining the reforming unit in an anaerobic state, and formic acid. And a system for supplying formate or glucose or an organic substance containing at least one component thereof.
By changing various types of the carbon source supplied as the organic substance, the time for developing the hydrogen generation ability can be changed. This utilizes the fact that the rate at which microorganisms take up a carbon source varies depending on the type of carbon source. For example, when galactose is used as the carbon source, it is possible to control the expression of hydrogen generation ability to be slower than when glucose is used as the carbon source. In addition, in addition to the carbon source supplied as an organic substance, it is also preferable to supply a nitrogen source and a mineral source shown as nutrient sources for microbial growth.

  The hydrogen generating ability expressing part has an anaerobic atmosphere imparting means. As the anaerobic atmosphere imparting means, means for heat-treating the contents in the reforming section, means for reducing the pressure in the reforming section, means for introducing an inert gas such as nitrogen gas or carbon dioxide gas into the reforming section, etc. They can be used in combination.

When the reforming part is depressurized to an anaerobic state, the reforming part is about 6.67 × 10 ² Pa or less, more preferably about 4.00 × 10 ² Pa or less, preferably about 1 to 60 minutes, preferably It is preferable to hold for about 5 to 60 minutes. The reforming section is preferably maintained in an anaerobic state even during the reforming reaction, and a method of sealing the reaction system with an inert gas such as nitrogen gas or carbon dioxide gas is usually used.

  If necessary, an appropriate reducing agent (for example, thioglycolic acid, ascorbic acid, cysteine hydrochloride, mercaptoacetic acid, thiolacetic acid, glutathione, sodium sulfide, etc.) is added to the contents in the reforming section to make anaerobic It is also possible to use a means for introducing such a reducing agent.

  The oxidation-reduction potential in the solution in the hydrogen generating ability developing part is preferably -100 mV to -500 mV, more preferably -150 mV to -400 mV, and further preferably -200 mV to -350 mV. The oxidation-reduction potential can be measured by, for example, ORP Electrodes manufactured by BROADLEY JAMES.

As an organic substance to be supplied to the reforming unit in order for the microorganisms to exhibit hydrogen generation ability, at least one selected from glucose, formic acid and formate can be used.
The glucose concentration is preferably 1 to 1000 mM, more preferably 1 to 300 mM, and even more preferably 10 to 250 mM, in terms of facilitating the expression of the hydrogen production capacity. To do.
As the formate, zinc formate, sodium formate, potassium formate, cesium formate, nickel formate, barium formate, calcium formate, ammonium formate and the like can be used. When using a formic acid or formate solution, these concentrations are preferably supplied so as to be 1 to 1000 mM, more preferably 1 to 500 mM, still more preferably 1 to 300 mM, Particularly preferred is 1 to 50 mM.

Examples of organic substances include sugars such as glucose, fructose, and molasses. When glucose is used, the amount of glucose is preferably an amount necessary for the number of cells to be about twice or more due to mitotic growth of microorganisms under anaerobic conditions. This mitotic growth can be easily known by performing a normal bacterial optical density measurement, for example, a measurement with spectrophotometer DU-800 manufactured by Beckman Coulter. In addition, inducible expression of formate dehydrogenase and hydrogenase includes necessary trace metal components (although metal components differ depending on the microorganism species used, iron, molybdenum, nickel, selenium, etc. are common). It is preferable.
When the microorganism growth medium contains glucose, the microorganism growth medium can be used as a glucose supply source.

  Induction of the hydrogen-producing ability of the microorganism in the hydrogen-producing ability-expressing part is performed at a temperature of about 20 ° C to 40 ° C, preferably about 25 ° C to 40 ° C. Moreover, pH can be performed in 5-10, Preferably it is 6-8 vicinity. The time for inducing the hydrogen-producing ability of the microorganism varies depending on the microorganism used, but is usually 0.5 to 24 hours.

  It is preferable that the microorganism culture part and the reforming part communicate with each other using at least one selected from piping, pumps, sensors, and valves. In addition, as described above, a separation unit can be interposed between the culture unit and the reforming unit.

The reforming section preferably has a mechanism for detecting the amount of liquid in the reforming section, and it is preferable to adjust the input amount of microorganisms from the culture section based on the detected value.
In the case where a microorganism that exhibits hydrogen generation ability in the culture apparatus of the present invention is used, for example, in an apparatus such as a hydrogen production apparatus, the above reforming unit can store and supply the microorganism that exhibits hydrogen generation ability. It is preferable to communicate with the microorganism reservoir. The microorganism storage unit preferably has a mechanism capable of supplying microorganisms to the hydrogen production apparatus continuously or intermittently.

In FIG. 1, the reforming tank 26 as a hydrogen generating ability developing unit is placed in an anaerobic state by an exhaust device (not shown), and then an organic substance supply device 27 uses a pump 42 and a flow meter 41. Add organics while controlling by. Microorganisms that express hydrogen generation ability in the reforming tank are sent to the microorganism storage tank 28 and can be stored until they are supplied to the reaction tank 2 of the continuous hydrogen generation apparatus described later as needed.
The above is a preferred example of the culture apparatus of the present invention.

FIG. 2 shows an example of a continuous hydrogen generation system including a microorganism culture apparatus connected to the hydrogen generation reaction section of the continuous hydrogen generator.
The hydrogen production apparatus of the present invention is preferably one in which the culture apparatus is connected to the hydrogen generation reaction section. In order to remove metabolites such as ethanol, acetic acid, and lactic acid, a part or all of the solution other than the microorganism is separated from the reforming vessel to the hydrogen generation reaction part.
Examples of the separation method include a method of reducing the amount of a culture solution containing a metabolite by a cross-flow filtration method using a filter, a method of separating a microorganism from a culture supernatant by centrifugation, and removing the culture supernatant. It is done. When removing the culture supernatant by centrifugation, it is preferable to disperse and suspend the microorganism after centrifugation in a new medium, wash it, and send it to the hydrogen generation reaction section for use. Further, in order to reduce the amount of metabolites brought into the hydrogen generation reaction part, it is preferable to repeat the above centrifugation and washing.
The above metabolites are known to adversely affect microbial hydrogen production. In the hydrogen production apparatus of the present invention, the amount of metabolites produced in the microorganism culture apparatus can be reduced as much as possible and delivered to the hydrogen production reaction section, so that hydrogen production by microorganisms can be performed stably for a long time. be able to.

  The culture apparatus including the microorganism culture part and the hydrogen production ability expression part in the hydrogen production apparatus is the same as described for the culture apparatus.

  The culture apparatus and the hydrogen production reaction part are preferably communicated by a mechanism capable of transporting microorganisms. As a mechanism capable of transporting microorganisms, an ordinary microorganism transport mechanism in which pumps, pipes and the like are appropriately combined can be used. The microorganism storage section of the culture apparatus is set to a positive pressure, and the microorganisms are placed in the hydrogen generation reaction section. Examples thereof include a mechanism for charging, a mechanism for introducing a microorganism from the culture apparatus by setting the inside of the hydrogen generation reaction section to a negative pressure, and the like.

  In FIG. 2, the microorganisms modified in the reforming tank 26 as described above are stored in the microorganism supply tank 28, and if necessary, the hydrogen generation reaction tank (hereinafter referred to as reaction) of the hydrogen generator by the power 14. (Also called tank). The microbial concentration when supplying to the reaction tank 2 is in the concentration range of 0.1 to 80% (w / w) (wet cell mass standard), preferably 10 to 70% (w / w) (wet cell) Body mass basis).

  A hydrogen production medium can be supplied to the reaction tank 2 from the medium tank 3. As the medium, it is preferable to use a medium different from the microorganism growth medium used in the culture section in terms of the ability of microorganisms to generate hydrogen, but the same medium can also be used. When the same medium as the microorganism growth medium is used as the hydrogen generation medium, the medium can be supplied from the medium tank 3 to the culture tank 24 of the culture apparatus. In this way, the hydrogen generator can be made compact.

The hydrogen generation reaction part is controlled at a temperature of 20 ° C. to 40 ° C., and preferably maintained at a temperature of 25 ° C. to 40 ° C.
The hydrogen generation reaction part has an anaerobic atmosphere imparting means. The anaerobic atmosphere imparting means is the same as that described for the anaerobic atmosphere imparting means in the hydrogen generating ability expressing part.

The hydrogen generation reaction section preferably communicates with a hydrogen generation organic substance (hereinafter also referred to as organic substrate) supply source that supplies an organic substance for hydrogen generation. The organic substance for hydrogen generation may be a compound such as a saccharide that is converted into formic acid in the intracellular metabolic pathway, or formic acid or formate. Here, the formate is a substance having a hydrocarboxyl group (chemical structural formula HCOO ⁻ ). Among them, formic acid, sodium formate, potassium formate, calcium formate, manganese formate, nickel formate, cesium formate, barium formate, ammonium formate and the like can be mentioned. Among these, formic acid, sodium formate, potassium formate, calcium formate, and ammonium formate are preferable from the viewpoint of solubility in water, and formic acid, sodium formate, and ammonium formate are preferable from the viewpoint of cost. Although the indirect supply method and the direct supply method can be used in combination, the direct supply method can be preferably used. And while controlling the solution containing the organic substance for hydrogen generation from the fuel tank 1 to the reaction tank 2 maintained in an anaerobic state using the pump 13 and the flow meter 12, the pH of the solution is in the range of 5-9. Can be supplied as When pH changes greatly, a pH adjusting solution can be supplied.
In this manner, by supplying an organic substance for hydrogen generation, a gas containing hydrogen, or a gas containing hydrogen, a metabolite, and the like are generated.

The hydrogen generation reaction section preferably has a means for flowing the liquid inside the reaction section in order to increase the area where the organic substance for generating hydrogen and the microorganisms come into contact with each other. As such means, there can be used a mixing blade for moving the contents of the reaction section, a device for spraying the contents, a device for dropping the contents provided inside the reaction tank, and the like.
The reforming tank 26, the organic substance supply device 27, the reaction tank 2, and the fuel tank 1 are selected to be materially stable depending on the characteristics of the solution, and part or all of the inside of the tank or tank Are preferably coated with a chemically stable material. For example, if an acidic solution is used, a tank made of an acid-resistant material such as an alloy can be used. The surfaces of the reforming tank 26 and the reaction tank 2 are preferably resistant to corrosion.

  When gas other than hydrogen gas is contained in the gas generated in the reaction section and it is desirable to remove it, a hydrogen concentration section (fixed section) is provided after the hydrogen generation reaction section, and components other than hydrogen gas Can be fixed. As a method for fixing components other than hydrogen gas, a conventionally known method can be used, and a fixing method using an absorbent using a liquid absorbent, a solid absorbent, or the like can be used. When removing carbon dioxide, a method of fixing the carbon dioxide by storing a liquid carbon dioxide absorbent in a hydrogen concentrating part can be used. As a liquid carbon dioxide absorbent, an ethanolamine solution or the like can be used.

  In FIG. 2, hydrogen gas can be concentrated by providing a hydrogen concentration tank 5. The valve 21 included in the hydrogen concentration tank 5 is a valve for replacing the thickener (absorbent), but there may be two or more replacement ports. Also, the absorbent regeneration system can be combined by combining a plurality of valves, tanks, and the like. When using a system that adsorbs at room temperature and regenerates at a high temperature, such as zeolite or amine-based solution, it is possible to regenerate the concentrating agent by using heat generated in the fuel cell section described later.

  The hydrogen production apparatus includes a hydrogen production amount detection unit, and is configured to be able to supply microorganisms from the culture device based on the detection value of the hydrogen production amount detection unit. As the hydrogen generation amount detection unit, a device capable of measuring the amount of generated hydrogen can be used, and for example, a gas flow meter can be used. In FIG. 2, the hydrogen generation amount can be measured by the flow meter 32, and the microorganisms can be supplied from the microorganism storage tank 28 to the reaction tank 2 based on the measured value.

  The supply amount of the solution containing the organic substance for hydrogen generation to the reaction tank is based on information such as the output of the fuel cell 6 described later, the flow meter 32 for detecting the hydrogen generation amount, the temperature of the reaction tank 2, the stirring state, etc. It can be controlled by adjusting. If the power generation output of the fuel cell 6 does not increase regardless of the increase in the output of the supply pump, it is considered that the ability of microorganisms to produce hydrogen has dropped, so the medium in the reaction tank 2 and a part of the microorganisms are extracted, It is preferable to supply microorganisms and a new medium from the medium tank and the culture apparatus. At that time, the amount of microorganisms supplied from the microorganism storage tank 28 to the reaction tank 2 is controlled.

  As described above, the cells in the reaction tank 2 and a part of the medium are transferred to the waste liquid tank 4 using the pump 18 while controlling the extraction amount based on information such as the hydrogen generation amount, temperature, and raw material supply amount. Extracted. A predetermined amount of medium is supplied from the medium tank 3 and a predetermined amount of microorganisms from the microorganism storage tank 28 are supplied to the reaction tank 2 using the pump 15 in accordance with the extracted amount. Fuel tank 1, reaction tank 2, and medium tank 3 use heat insulating materials such as polyurethane foam and glass wool to control the amount of heat transfer, and the temperature is controlled to about 20 to 45 ° C using a heat exchanger. It is preferable that

Hereinafter, a hydrogen production apparatus capable of controlling the hydrogen production amount and a system including a fuel cell will be described.
FIG. 3 shows, as an example, a configuration diagram of a fuel cell system using a hydrogen production apparatus capable of controlling the hydrogen production amount.
While being supplied from the tank 52 containing the organic substrate of FIG. 3 by the organic substrate supply pump 61, it is supplied to the hydrogen generation reaction unit 51. The hydrogen generation reaction unit 51 may include a reaction solution including a microorganism having a formate dehydrogenase gene and a hydrogenase gene and a medium component (hydrogen generation medium). By supplying the organic substrate to the hydrogen generation reaction unit 51, the microorganisms in the reaction solution produce gas containing hydrogen.

  The gas produced by the microorganisms passes through the condenser 55, is separated into hydrogen-rich gas by the gas separation device 56, and is supplied to the fuel electrode of the fuel cell 54. The fuel cell 54 can generate power from hydrogen gas supplied to the fuel electrode and oxygen in the air supplied to the air electrode.

The amount of gas produced can be controlled by controlling the amount of organic substrate supplied by the organic substrate supply pump 61. Furthermore, the amount of power generated by the fuel cell 54 can be controlled by controlling the supply amount of the organic substrate.
Formic acid concentration, temperature, pH, microorganism concentration can be confirmed by attaching necessary sensors to various sensors 67 so that the formic acid concentration, temperature, pH, microorganism concentration in the reaction solution can be grasped. A hydrogen production apparatus capable of setting the conditions of pH, microbial concentration is obtained. About stirring, the hydrogen production apparatus which can set the conditions of the rotation speed of a stirring apparatus is obtained by attaching the stirring apparatus 60 to a reaction container, rotating, and grasping | ascertaining the rotation speed.

  In addition, when the above hydrogen production apparatus is used as a fuel cell system, hydrogen is controlled by controlling at least one hydrogen production condition selected from formic acid concentration conditions, temperature conditions, pH conditions, microbial concentration conditions, and stirring conditions. It is possible to obtain a fuel cell system including a hydrogen production apparatus capable of controlling the production amount and at the same time controlling the power generation amount as the fuel cell system.

  Details of the formic acid concentration condition, temperature condition, pH condition, microbial concentration condition, and stirring condition will be described below as means for controlling the hydrogen production amount and the power generation amount of the fuel cell.

  The formic acid concentration condition is controlled by measuring the formic acid ion concentration in the reaction vessel. Examples of the method for measuring the formic acid concentration include a method using liquid chromatography, and a method using an ultrasonic liquid concentration meter that measures ultrasonic waves and conductivity. In the present invention, in order to observe the change over time in the solution, the measurement is performed by a method using an ultrasonic densitometer, but a method using liquid chromatography can also be used in combination. Increasing the formic acid concentration increases the hydrogen production, and decreasing the formic acid concentration decreases the hydrogen production. In addition, when the concentration of formic acid exceeds a certain level, it becomes difficult to control the amount of hydrogen produced. The range of the formic acid concentration that can control the hydrogen production amount is preferably 0 to 250 mM, and more preferably 10 to 100 mM in that the linear relationship between the hydrogen production amount and the formic acid concentration can be obtained. A method for increasing the formic acid concentration is performed by adding an organic substrate, and a method for decreasing the formic acid concentration is performed by a method of adding a medium component or the like.

  The reaction temperature condition is controlled by measuring the temperature in the reaction vessel. Examples of the method for measuring the temperature include a method using a general resistance temperature sensor. Increasing the reaction temperature increases the hydrogen production, depending on the microorganism species used, and decreasing the temperature decreases the hydrogen production. In general, when a microorganism that grows at room temperature is used, the temperature range in which hydrogen production can be controlled is preferably 20 ° C to 45 ° C, more preferably 30 ° C to 40 ° C in terms of the life of the microorganism. To preferred. As a method for raising the reaction temperature, a method of heating a hydrogen production apparatus using thermal energy obtained by a fuel cell is performed. Although methods for lowering the reaction temperature include air cooling and water cooling, the water cooling method is used in terms of easy control.

  The pH condition is controlled by measuring the pH in the reaction solution. As a method for measuring pH, a value is detected using a general pH sensor or the like. The pH value depends on the microorganism species to be used, but as the difference between the optimum pH range and the pH value in the reaction solution decreases, the hydrogen production increases, and as the difference increases, the hydrogen production increases. Decrease. In general, although the optimum value varies depending on the microorganism species to be used and the buffer component in the reaction solution, the optimum pH value is often present at 4.0 to 8.0, and more preferably 5.5 to 7 0. As a method for increasing the pH, a method of adding an alkaline solution can be used. As a method for lowering the pH, a method of adding an organic substrate is used. Examples of the alkaline solution include aqueous solutions of lithium hydroxide, sodium hydroxide, potassium hydroxide, rubidium hydroxide, cesium hydroxide, ammonia, and the like. Among these, sodium hydroxide is preferably used from the viewpoint of cost and ammonia water can be used as a nitrogen source.

  The microorganism concentration condition is controlled by measuring the microorganism concentration in the reaction solution. Examples of the method for measuring the microbial concentration include a method using a turbidity sensor, a method for calculating from the absorbance, and a method for applying an electric field to a part of the reaction solution and measuring the degree of polarization of the cells in that part. In the present invention, in order to observe the change with time in the solution, although measurement is performed with a turbidity sensor, a method of calculating from the absorbance can be used together, but the measurement method is not limited to these. Increasing the microbial concentration increases the hydrogen production, and decreasing the microbial concentration decreases the hydrogen production. As a method of increasing the microorganism concentration, a method of introducing microorganisms from the microorganism supply port 66 is used. As a method for reducing the microorganism concentration, a method of supplying a medium from the medium component supply pump 62 into the reaction solution is used.

  Stirring conditions are controlled by setting the rotation speed of the stirring device 60 in the reaction solution. Although various control methods, such as a method of measuring not only the rotation speed but also the torque of the motor 59 and the power consumption of the stirring apparatus 60, can be cited as the stirring conditions, FIG. 3 shows an apparatus that uses the rotation speed. When the rotation speed of the stirring device 60 is increased, the hydrogen production amount is increased, and when the rotation speed of the stirring device 60 is decreased, the hydrogen production volume is decreased. The rotation speed of the stirring device 60 can be adjusted by adjusting the motor 59. A magnetic stirring device is also preferably used because maintenance is easy.

  The amount of hydrogen generation can be controlled by controlling according to at least one hydrogen production condition selected from the formic acid concentration condition, temperature condition, pH condition, microorganism concentration condition, and stirring condition described above. In addition, by controlling the hydrogen production amount in this way, the power generation amount of the fuel cell can also be controlled.

  The hydrogen production amount can also be controlled by combining two or more of the hydrogen generation conditions shown above. Among these, a combination of formic acid concentration conditions and microbial concentration conditions, and a combination of microbial concentration conditions and stirring conditions are preferred, and a method of simultaneously controlling these is preferred. By simultaneously controlling the formic acid concentration condition and the microbial concentration condition, and the microbial concentration condition and the stirring condition, it becomes possible to more easily control the hydrogen production amount.

  As a means for supplying the organic substrate to the reactor (hydrogen generation reaction unit) 51, a method of supplying the organic substrate while spraying using the device 57 for spraying the organic substrate can be used. By performing the spraying, the organic substrate can be distributed and supplied, the reaction area can be increased, and the production rate of the gas containing hydrogen can be increased accordingly. In particular, when formic acid is used as the organic substrate, it is possible to reduce the local acid load on the microorganisms in the reaction solution by spraying. As a result, it is preferable because microbial life is improved. Examples of the means for spraying include a method using pressure and a method using ultrasonic waves. When a method using pressure is used as a means for spraying, it is necessary to use an inert gas such as nitrogen gas in order to maintain an anaerobic atmosphere in the reactor 51. From this point, the means for spraying is preferably a method using ultrasonic waves.

By using the condenser 5 in the vicinity of the discharge port of the gas containing hydrogen generated from the reactor 51, the amount of vapor contained in the gas containing hydrogen produced by the microorganism can be reduced. The cooling method may be air cooling, but is preferably water cooled in order to enhance the effect. As a result, the vapor contained in the generated gas forms condensation, droplets, and a liquid film in the piping of the system from the reactor 51 to the fuel cell, and the supply of the generated hydrogen to the fuel cell tends to be delayed. Improved.
The step of generating a gas containing hydrogen gas from the supply of the organic substrate is preferably performed in a constant temperature atmosphere in the thermostatic chamber 53.

The organic substrate can be replenished from the organic substrate supply port 63.
The gas separation device 56 can separate a hydrogen-rich gas from a gas mainly composed of hydrogen and carbon dioxide generated in the reactor 51. As a separation method, a general method such as a membrane separation method or an adsorption method is used.

  A tank 58 containing medium components, a medium component supply pump 62, a medium component supply port 64, and a reaction solution discharge valve 65 can be installed. Here, when hydrogen is continuously produced, a method is used in which medium components are added from the medium component supply pump 62 continuously or semi-continuously, and the reaction solution is withdrawn from the reaction solution discharge pump 65 at the same flow rate. Microorganisms can also be supplied from the microorganism supply port 66. The medium components in the tank 58 containing the medium components can be supplied from the medium component supply port 64.

  In order to generate hydrogen in microorganisms in the reactor, formic acid and formate are supplied continuously or intermittently (direct supply method), or sugars that are converted to formic acid in the intracellular metabolic pathway (eg glucose , Fructose, mannose, galactose, etc.) (indirect supply method). A direct supply method and an indirect supply method can be used in combination.

Here, the formate is a substance having a hydrocarboxyl group (chemical structural formula HCOO ⁻ ). Among them, formic acid, sodium formate, potassium formate, calcium formate, manganese formate, nickel formate, cesium formate, barium formate, ammonium formate and the like can be mentioned. Among these, formic acid, sodium formate, potassium formate, calcium formate, and ammonium formate are preferable from the viewpoint of solubility in water, and formic acid, sodium formate, and ammonium formate are preferable from the viewpoint of cost. The concentration of formic acid and formate to be supplied does not increase the volume of the reaction solution in the reactor 51, so that the concentration is close to the saturation concentration of each substance. Formic acid which is a liquid raw material with high purity is more preferable in terms of handling.
As a reaction solution condition, it is necessary to use a culture solution in a reduced state. According to anaerobic conditions, the oxidation-reduction potential is −100 mV (millivolt) to −500 mV, preferably −200 mV to −500 mV.

It is preferable to use a hydrogen generation reaction solution having a microorganism concentration of 10% (w / w) to 90% (w / w) (wet cell mass standard). From the viewpoint of increasing the viscosity of the reaction solution, the microorganism concentration is preferably 10% (w / w) to 80% (w / w) (wet cell mass basis). More preferably, from the viewpoint of the amount of hydrogen generated per unit volume, the microbial concentration is preferably 20% (w / w) to 80% (w / w) (wet cell mass basis). By producing hydrogen in this microbial concentration range, it is possible to obtain a hydrogen production rate of 50 L (H ₂ ) / hr / L (reaction volume) or more, which is the hydrogen productivity necessary for practical use.

  Examples of the medium components that can be contained in the reaction solution used in the reactor 51 include a normal nutrient medium containing a carbon source, a nitrogen source, a mineral source, and the like. Examples of the carbon source include glucose, fructose, molasses, etc., and examples of the nitrogen source include inorganic nitrogen sources such as ammonia, ammonium salts, and nitrates, and organic nitrogen sources such as urea, amino acids, and proteins. Each can be used alone or in combination. Both inorganic and organic forms can be used in the same manner. Further, as the mineral source, for example, potassium monohydrogen phosphate, magnesium sulfate or the like mainly containing K, P, Mg, S and the like can be used. In addition, nutrients such as various vitamins such as peptone, meat extract, yeast extract, corn steep liquor, casamino acid, biotin, thiamine and the like can be added as necessary.

  When generating hydrogen, it is preferable to add an antifoaming agent to the reaction solution. About an antifoamer, a well-known thing is used. Specifically, silicone-based and polyether-based antifoaming agents are used.

  As the material of the reactor 51, acid-resistant metals, glass and plastics treated with acid-resistant coating can be preferably used. In addition, since the reactor 51 uses anaerobic microorganisms, a structure that does not allow oxygen to permeate into the reactor 51 is preferable, and it is recommended that the rotating portion of the stirring portion has a high sealing property using packing or the like. .

In the conventional technology, when hydrogen produced by a hydrogen production device is generally used as a fuel for a polymer electrolyte fuel cell, a system for removing carbon monoxide (CO converter, CO remover, etc.) is used. , CO must be kept below 10ppm. In the hydrogen production apparatus using the microorganism of the present invention, a gas mainly composed of hydrogen and carbon dioxide is produced, and basically no carbon monoxide is produced. Therefore, in the method of the present invention, it is not necessary to install an apparatus for removing CO.
In addition, a conventional hydrogen generator using city gas requires a reforming temperature of 600 ° C or higher, and a reforming method using methanol requires a reforming temperature of several hundreds of ° C. The temperature of the reaction vessel of the invention can be used at room temperature. Further, although it usually takes time to start up and finish the conventional reformer, the method of the present invention can be easily handled until the production of hydrogen and can shorten the time, and is preferable as a fuel cell system.

Furthermore, the electric power generated in the fuel cell can be connected to the equipment system via the power conversion system 7 as shown in FIG. The gas discharged from the fuel cell generates heat through the combustion catalyst 9 and is used as part of a heat source for keeping the fuel tank 1, the reaction tank 2, the medium tank 3, and the hydrogen concentrating tank 5 as necessary. Can do.
From these points as well, the fuel cell system using the hydrogen production method of the present invention does not generate CO, so there are few problems with deterioration of the fuel cell, and a reformer that requires a high temperature as a hydrogen supply method. It can be seen that the system is superior in that it does not require a system and that hydrogen can be produced simultaneously with the supply of the organic substrate.

  According to the present invention, microorganisms having hydrogen-producing ability can be continuously cultured to develop hydrogen-producing ability, and once a seed microorganism is supplied, useful microorganisms are produced continuously for a long period of time. it can. In addition, it becomes possible to continuously supply a microorganism that exhibits hydrogen generation ability to a hydrogen generator, and it is possible to continuously generate hydrogen using the microorganism.

  The hydrogen production apparatus of the present invention and the fuel cell system including the hydrogen production apparatus can control the production amount of gas containing hydrogen generated in the reactor 51 by controlling the supply amount of the organic substrate. Can also control the amount of power generated by the fuel cell. Further, by using formate and formic acid as the organic substrate, it is difficult to produce a fermentation residue, and since the main product is a gas, separation from the reactor 51 is easy. It is characterized in that hydrogen production can be easily performed.

Hereinafter, the present invention will be specifically described by way of examples, but the present invention is not limited thereto.
Example 1
The hydrogen generator used in Example 1 is shown in FIG.
In the microbial culture medium tank 23, water (1000 ml), glucose (20 mM), tryptone peptone (1%), sodium molybdate (10 μM), sodium selenite (10 μM), disodium hydrogen phosphate (26.5 mm), sodium dihydrogen phosphate (73.5 mM), yeast extract (0.5%) and sodium sulfide (2 mM) previously sterilized by heating at 120 ° C. for 10 minutes is stored and supplied to the culture tank. Only at the first start-up, Escherichia coli W strain (ATCC9637) is put into the culture tank, and the medium is supplied so that the cell density is 3 g-wet cell / liter (cell optical density OD ₆₁₀ = about 1.5). did. Then, sterilized air was sent and aerated. After culturing at 37 ° C for 12 hours, the necessary amount of cells and culture solution in the culture tank is determined from the liquid volume sensor installed in the tank in the hydrogen production capacity expression tank, and the supply volume from the flow meter Was determined and sent to a hydrogen generation capacity expression tank using a pump. The culture solution discharged from the culture tank was once separated from the culture medium and washed in a bacterial cell washing tank (not shown), mixed with a new culture medium, and then sent to the hydrogen production capacity expression tank. . At that time, the oxygen concentration in the tank was reduced by using an exhaust device in order to make the hydrogen generating capacity expressing tank anaerobic. At that time, the oxidation-reduction potential of the contents in the hydrogen generation capacity expression tank was −250 mV. And in order to express hydrogen production ability, sodium formate was supplied so that it might become about 50 mM, and hydrogen production ability was made to express in a microbial cell. The time for inducing the hydrogen generation ability was 6 hours. Microorganisms that expressed hydrogen generation ability were stored in a microorganism storage tank.

  In this continuous microbial hydrogen production capacity expression device, a water level sensor is installed in each of the medium tank, culture tank, microbial cell washing tank, reforming container (hydrogen production capacity expression tank), and microbial storage tank, and a flow meter and pump The solution was fed using Further, the inside of each tank was controlled at a temperature of 37 ° C. and a pH of about 6-7.

In the hydrogen generation medium tank 3, water (1000 ml), tryptone peptone (1%), sodium molybdate (10 μM), sodium selenite (10 μM), disodium hydrogen phosphate ( 26.5 mM), sodium dihydrogen phosphate (73.5 mM), and yeast extract (0.5%) were preliminarily heat-sterilized at 120 ° C. for 10 minutes and stored.
In the reaction tank 2, a medium for hydrogen generation is supplied from the medium tank 3 using the pump 15, and further, the cells are supplied from the microorganism storage tank, maintained at a temperature of about 37 ° C. and a pH of 6 to 7, and kept in an anaerobic state. Then, the formic acid aqueous solution was fed from the fuel tank 1 while being measured by the flow meter 12 using the pump 13. Stirring was performed with the stirrer 8 to improve the contact frequency between the cells and formic acid.

The mixed gas containing generated hydrogen passed through a pipe having a flow meter 32, and hydrogen was concentrated by adsorbing carbon dioxide to diethanolamine in the hydrogen concentration tank 5.
Concentrated hydrogen is sent to the anode of the fuel cell unit 6 using the pump 45, and air taken in by the pump 20 from the air intake port 19 is removed by the filter 10 and then sent to the cathode. These were used to generate electricity. The generated power is connected to the equipment / system by an inverter 7 or the like.

In the reaction tank 2, since it was necessary to replenish the cells and replace the medium along with the generation of hydrogen gas, the culture solution in the reaction tank 2 was extracted to the waste liquid tank 4 by the pump 18.
By repeating the above operations, it was possible to continuously generate hydrogen. As a result of measuring the amount of gas generated by continuously supplying an aqueous formic acid solution with a concentration of 26 mol (mol) / L (liter) at a feed rate of 28 mL / hr, it was about 32 L (gas) / hr, 120 hours It was possible to perform continuous gas production. In the analysis by gas chromatography, the main components of the product gas were hydrogen and carbon dioxide, which were produced in almost the same volume. The hydrogen evolution rate was stably 50-55 L (H ₂ ) / hr / L (reaction volume) during the experimental time.

  From the results of Example 1, it has been clarified that the hydrogen required for the fuel cell is generated at a stable rate over several days by using the hydrogen production apparatus provided with the culture apparatus of the present invention.

Examples 2-4
A method for producing hydrogen using a microorganism using Escherichia coli W strain (ATCC 9637).
This strain was added to 500 ml (milliliter) of a culture solution having the composition shown in Table 1 below, followed by shaking culture (preculture) overnight at 37 ° C. under aerobic conditions.

  Next, a part of the culture solution which has been subjected to overnight shaking culture (pre-culture) under anaerobic conditions is collected, added to the culture solution having the composition shown in Table 2 below containing sodium formate, and shaken at 37 ° C. for 12 hours. Culture (main culture) was performed.

Subsequently, the main culture solution was centrifuged (5000 rpm, 15 minutes), and the supernatant was removed to obtain a microorganism having a hydrogen production function.
The microorganism was separated by centrifugation and then suspended in 500 ml (milliliter) of a hydrogen generation medium under the reduced state shown in the composition of Table 3 below.

A schematic diagram of the apparatus used in this example is shown in FIG.
The hydrogen generation reaction solution containing the microorganisms prepared above was injected into the reactor 51, and the hydrogen generation medium shown in Table 3 was injected into the tank 58 containing the medium components. In addition, 26 M (molar mol / liter) formic acid was prepared in the tank 52 containing the organic substrate.
The reaction solution temperature was 37 ° C., the pH of the solution was 6.0, the microorganism concentration was about 40% (wet cell mass standard), and the stirring speed in the reaction solution was 800 rpm.

The organic substrate supply pump 61 was used to change the supply rate of the organic substrate, and the amount of hydrogen production gas generated by supplying to the reactor 51 and the amount of power generated in the fuel cell were measured.
The supply rate was 10 ml / hr (milliliter / hour) (Example 2), 21 ml / hr (milliliter / hour) (Example 3), and 42 ml / hr (milliliter / hour) (Example 4). The result is shown in FIG.

  FIG. 5 shows the correlation between the supply rate of the organic substrate, the hydrogen production amount, and the power generation amount in the fuel cell system. It became clear that the amount of hydrogen produced and the amount of power generated by the fuel cell can be controlled by controlling the supply rate of the organic substrate.

Example 5
A microorganism having hydrogen-producing ability was obtained in the same manner as in Example 2, and the obtained microorganism was suspended in a hydrogen-generating medium to obtain 500 ml (milliliter) of a hydrogen-generating reaction solution.
The formic acid was supplied to the hydrogen production apparatus with the organic substrate supply pump 61, and the hydrogen production rate and the amount of power generated in the fuel cell were measured while adding the formic acid to the following concentration in the reaction solution.

Except for changing the formic acid concentration in the reactor to 10 mM (millimolar), 30 mM (millimolar), 50 mM (millimolar), the temperature of the reaction solution in the reactor is 37 ° C., the pH is 6.0, and the microbial concentration is 40% (w / w) (wet cell mass standard), stirring was controlled by the number of revolutions of the stirrer, and the hydrogen production rate and power generation were measured at 800 rpm.
The result is shown in FIG.

  FIG. 6 shows the correlation between the concentration of the organic substrate (formic acid), the hydrogen production amount, and the power generation amount in the fuel cell system. By controlling the formic acid concentration in the reaction solution of the reactor, it became clear that there is a correlation between the hydrogen production amount and the power generation amount of the fuel cell.

Example 6
The temperature of the reaction solution in the reactor was changed to 30 ° C., 37 ° C., and 44 ° C., formic acid concentration: 30 mM, pH = 6.0, microorganism concentration: 40% (w / w), and stirring rotation speed: 800 rpm Was used to measure the hydrogen production rate and the amount of power generated by the fuel cell in the same manner as in Example 5.
The results are shown in FIG.

  FIG. 7 shows the correlation between the temperature of the reaction solution in the reactor, the hydrogen production amount, and the power generation amount in the fuel cell system. By controlling the temperature in the reaction solution of the reactor, it became clear that there is a correlation between the hydrogen production amount and the power generation amount of the fuel cell.

Example 7
The pH of the reaction solution in the reactor was changed to 5.5, 6.0, and 6.5, formic acid concentration: 30 mM, temperature: 37 ° C., microorganism concentration: 40% (w / w), stirring rotation speed: 800 rpm Using these conditions, the hydrogen production rate and the amount of power generated by the fuel cell were measured in the same manner as in Example 5.
The results are shown in FIG.

  FIG. 8 shows the correlation between the pH of the reaction solution in the reactor, the hydrogen production amount, and the power generation amount in the fuel cell system. It has been clarified that there is a correlation between the hydrogen production amount and the power generation amount of the fuel cell by controlling the pH in the reaction solution of the reactor.

Example 8
The reaction solution in the reactor was changed to a microbial concentration of 30%, 40%, and 50%, using formic acid concentration: 30 mM, temperature: 37 ° C., pH = 6.0, stirring rotation speed: 800 rpm. In the same manner as in Example 5, the hydrogen production rate and the power generation amount of the fuel cell were measured.
The results are shown in FIG.

  FIG. 9 shows the correlation between the microbial concentration of the reaction solution in the reactor, the hydrogen production amount, and the power generation amount in the fuel cell system. By controlling the microbial concentration in the reaction solution of the reactor, it became clear that there is a correlation between the hydrogen production amount and the power generation amount of the fuel cell.

Example 9
The stirring force of the reaction solution in the reactor was controlled by the rotation speed of the stirring device. The examples were prepared by changing the rotation speed of the stirring device to 400 rpm, 800 rpm, and 1200 rpm, and using the conditions of formic acid concentration: 30 mM, temperature: 37 ° C., pH = 6.0, and microorganism concentration: 40% (w / w). 5 was used to measure the hydrogen production rate and the amount of power generated by the fuel cell.
The results are shown in FIG.

  FIG. 10 shows the correlation between the rotation speed of the stirring device in the reactor, the hydrogen production amount, and the power generation amount in the fuel cell system. It became clear that there was a correlation between the hydrogen production amount and the power generation amount of the fuel cell by controlling the rotation speed of the stirring device in the reactor.

  According to Examples 5 to 10, the amount of hydrogen produced from the reaction vessel is controlled by any one of formic acid concentration, temperature, pH, microbial concentration, and stirring force. It has been found that it is possible to control the amount of power generated by a fuel cell using hydrogen produced by using this as fuel gas.

  The culture apparatus of the present invention and the hydrogen generator including the same can generate hydrogen continuously using microorganisms, and are applied to systems that require hydrogen continuously, such as homes and office buildings. It can be used for a fuel cell cogeneration system, a fuel cell for a distributed power source widely installed in a demand area network, and the like.

Claims (9)

A microorganism culture part communicating with a gas source containing oxygen and a nutrient source supply part, and an organic substance that communicates with the culture part and contains glucose and formic acid or formate to express hydrogen generation ability to the microorganism An apparatus for culturing microorganisms having an organic substance supply source and an anaerobic atmosphere imparting means for microorganisms having an anaerobic condition under anaerobic conditions is connected to the hydrogen production reaction section.
The hydrogen production reaction unit, the formic acid or formate salt material feed portion communicating with supply via a spraying device,
Between the culture part and the hydrogen production ability expression part, a separation part of the cultured microorganism and the culture solution is provided,
Between the hydrogen generating ability expressing part and the hydrogen generating reaction part, there is provided a separating part for the microorganism that has expressed hydrogen generating ability and the culture solution.
This is a continuous hydrogen production system. The hydrogen production apparatus according to claim 1, wherein the microorganism has a formate dehydrogenase gene and a hydrogenase gene. The hydrogen production apparatus according to claim 1 or 2 , wherein the production amount of hydrogen is controlled by the supply amount of formic acid or formate from the raw material supply unit to the hydrogen production reaction unit. Production of hydrogen, the concentration condition of formic acid or formate salt of the hydrogen production reaction unit, temperature conditions, pH conditions, to claim 1 or 2 is controlled by at least one of the hydrogen generating conditions selected from microorganisms density conditions and stirring conditions The hydrogen production apparatus as described. Hydrogen production reaction unit comprises a hydrogen generation amount detection unit, according to consists of any one of claims 1-4 consisting so that it can supply the microorganisms from the culture device based on the detection value of the hydrogen generation amount detector Hydrogen production equipment. The hydrogen production apparatus according to any one of claims 1 to 5 , wherein the hydrogen generation reaction unit includes a condenser for condensing water vapor contained in the discharged gas. A system comprising the hydrogen production apparatus according to any one of claims 1 to 6 and a fuel cell using hydrogen generated from the hydrogen production apparatus as a fuel gas. The system according to claim 7 , wherein the amount of power generation in the fuel cell is controlled by the amount of formic acid or formate supplied from the raw material supply unit to the hydrogen generation reaction unit. The amount of power generation in the fuel cell is controlled by any one hydrogen generation condition selected from a concentration condition, a temperature condition, a pH condition, a microorganism concentration condition, and a stirring condition of formic acid or formate in the hydrogen generation reaction section. 8. The system according to 7 .

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