JP2005323515A - Method for producing hydrogen by microorganism - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing hydrogen, capable of producing the hydrogen with high energy conversion efficiency and capable of making facilities small as a whole. <P>SOLUTION: This method for producing the hydrogen comprises culturing anaerobic bacteria having ability of forming the hydrogen by using an organic acid as a substrate, or facultative anaerobic bacteria having the ability, in a culture medium containing the organic acid, so as to make the bacteria form the hydrogen. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a hydrogen production method, and more particularly to a hydrogen production method using microorganisms.

  Since hydrogen does not release carbon dioxide during combustion, it is expected to be widely used as a clean fuel. For example, it is listed as one of the next-generation influential energy sources, starting with supplying hydrogen to fuel cells and converting it into electrical energy.

  Conventionally, hydrogen has been produced by reforming fossil fuels such as naphtha and natural gas or by electrolysis of water. However, since these production methods consume fossil fuels, there is a problem that the production process causes pollution of the global environment. For this reason, research on clean and efficient hydrogen production technology using microorganisms is underway as a technology useful for improving the global environment.

  There are roughly two types of hydrogen production methods using microorganisms, one is light-dependent and the other is light-independent. The former method uses a photosynthetic bacterium that produces hydrogen in the process of assimilating organic matter under light irradiation. The latter includes a method utilizing an anaerobic bacterium or facultative anaerobic bacterium (for example, a genus Clostridium) having the ability to generate hydrogen using sugar as a substrate.

JP 2002-272491 A Japanese Unexamined Patent Publication No. 7-31998 JP-A-8-294396 Japanese Patent Laid-Open No. 8-308591

  However, in the light-dependent hydrogen production method, it is necessary to continuously irradiate light energy when culturing microorganisms. In addition, it is difficult to irradiate the deep part of the culture tank with light having a light intensity sufficient for photosynthesis, and hydrogen is generated only at and near the surface of the culture tank. Need. That is, the light-dependent hydrogen production method has a problem that the energy efficiency is poor and a problem that the entire facility is enlarged because a vast culture facility is required.

  On the other hand, in the photo-independent hydrogen production method, bacteria produce hydrogen by fermentation instead of photosynthesis, so the photo-independent hydrogen production method has high energy efficiency and does not require extensive culture facilities. However, these bacteria produce organic acids as by-products when they assimilate sugars to produce hydrogen. Therefore, this hydrogen production method has a problem that the organic acid as a by-product needs to be treated by a wastewater treatment facility or the like provided on the downstream side of hydrogen fermentation, and the entire equipment is enlarged.

  Therefore, in order to increase the utility value of hydrogen as clean energy, hydrogen production method using photosynthetic bacteria and hydrogen production method using anaerobic bacteria or facultative anaerobic bacteria having the ability to generate hydrogen using sugar as a substrate It is necessary to overcome the above problems.

  That is, an object of the present invention is to provide a hydrogen production method capable of producing hydrogen with high energy conversion efficiency and miniaturizing the entire facility.

  The present inventors paid attention to an organic acid generated as a by-product when assimilating sugars to generate hydrogen, and as a result of intensive research to solve the above problems, hydrogen was generated using an organic acid as a substrate. We have found that there are competent anaerobic bacteria or facultative anaerobic bacteria. As a result of further earnest studies, the present inventors have found that the problems of the conventional techniques can be solved by the following invention, and have completed the present invention.

  That is, the present invention is characterized by culturing an anaerobic bacterium or facultative anaerobic bacterium having an ability to generate hydrogen using an organic acid as a substrate in a medium containing the organic acid, and causing the bacterium to generate hydrogen. A method for producing hydrogen is provided.

  According to the method for producing hydrogen according to the present invention, since an organic acid is contained in the medium, the bacterium produces hydrogen using the organic acid as a substrate. For this reason, it becomes possible to produce | generate hydrogen with high energy conversion efficiency. Further, unlike the case of culturing by light irradiation, a vast culture facility is not required. Furthermore, since an organic acid is not unnecessary, a wastewater treatment facility for treating the organic acid is not required.

  In the hydrogen production method, the organic acid is preferably composed of at least one selected from the group consisting of lactic acid, acetic acid, propionic acid, citric acid, succinic acid, and malic acid. Compared with other organic acids, the above-mentioned organic acid has a feature that it is rapidly taken up and used in the metabolic pathway of microorganisms. The organic acid is not a special organic acid, but an organic acid contained in general organic waste. Other organic acids that can be used as a hydrogen source include organic acids related to the TCA cycle, formic acid, butyric acid, and isobutyric acid. An acid is preferred.

  The present invention also includes culturing an anaerobic bacterium or facultative anaerobic bacterium having an ability to generate hydrogen using an organic acid as a substrate in an organic waste liquid containing an organic acid, and causing the bacterium to generate hydrogen. A hydrogen production method is provided.

  According to the method for producing hydrogen according to the present invention, since an organic acid is contained in the organic waste liquid, the bacterium produces hydrogen using this organic acid as a substrate. For this reason, it becomes possible to produce | generate hydrogen with high energy conversion efficiency. Further, unlike the case of culturing by light irradiation, a vast culture facility is not required. Furthermore, since an organic acid is not unnecessary, a wastewater treatment facility for treating the organic acid is not required. In addition, according to the method for producing hydrogen of the present invention, the organic waste liquid that is no longer needed is purified, and at the same time becomes a hydrogen generation source, so that the organic waste liquid can be effectively used.

  Here, the organic waste liquid is preferably an organic residue generated during the production process of shochu. More than 500,000 tons of organic residues generated in the manufacturing process of shochu are generated annually in Japan. However, this organic residue contains a large amount of water and is difficult to treat, and most of the organic residue has been dumped into the ocean. On the other hand, this organic residue generally contains a large amount of organic acid. Therefore, by culturing the bacterium according to the present invention in this organic residue, it is possible to process the organic residue that has been difficult to process and to produce hydrogen more efficiently.

  The present invention further provides an anaerobic bacterium or facultative anaerobic bacterium having the ability to generate hydrogen and an organic acid using sugar as a substrate, and an anaerobic bacterium or facultative anaerobic having the ability to generate hydrogen using an organic acid as a substrate. Provided is a method for producing hydrogen, characterized by culturing a sexual bacterium in a medium containing sugar to cause both bacteria to produce hydrogen.

  According to this hydrogen production method, an anaerobic bacterium or facultative anaerobic bacterium having an ability to produce hydrogen using an organic acid as a substrate, and an anaerobic bacterium or facultative bacterium having an ability to produce hydrogen and an organic acid using a sugar as a substrate. When the anaerobic bacteria are cultured together, hydrogen and an organic acid are generated using the sugar contained in the medium as a substrate, and further hydrogen is generated using the generated organic acid as a substrate. For this reason, it becomes possible to produce | generate hydrogen with high energy conversion efficiency. Further, unlike the case of culturing by light irradiation, a vast culture facility is not required. Furthermore, according to this hydrogen production method, an organic acid is not required. That is, even when an organic acid is generated by generating hydrogen using a sugar as a substrate, hydrogen is generated using the organic acid as a substrate and the organic acid is processed. For this reason, the waste water treatment facility for processing an organic acid becomes unnecessary. Furthermore, according to this hydrogen production method, not only anaerobic bacteria or facultative anaerobic bacteria capable of generating hydrogen using an organic acid as a substrate, but also anaerobic capable of generating hydrogen using a sugar as a substrate. Hydrogen is also produced by culturing sexual or facultative anaerobic bacteria. For this reason, compared to when anaerobic bacteria or facultative anaerobic bacteria having the ability to generate hydrogen using an organic acid as a substrate can be cultured alone, the amount of hydrogen produced can be increased more efficiently. It is possible to produce hydrogen.

  The present invention further comprises culturing an anaerobic bacterium or facultative anaerobic bacterium having the ability to produce hydrogen and an organic acid using sugar as a substrate in a first medium containing sugar, In the second medium containing the generated organic acid, an anaerobic bacterium or facultative anaerobic bacterium capable of generating hydrogen using the organic acid as a substrate is cultured to generate hydrogen in the bacterium. A method for producing hydrogen is provided.

  According to this hydrogen production method, hydrogen and organic acid are produced by culturing anaerobic bacteria or facultative anaerobic bacteria having the ability to produce hydrogen and organic acids using sugar as a substrate. Further, hydrogen is further generated using the organic acid generated at this time as a substrate. For this reason, it becomes possible to produce | generate hydrogen with high energy conversion efficiency. Further, unlike the case of culturing by light irradiation, a vast culture facility is not required. Furthermore, according to this hydrogen production method, an organic acid is not required. That is, even when an organic acid is generated by generating hydrogen using a sugar as a substrate, hydrogen is generated using the organic acid as a substrate and the organic acid is processed. For this reason, the waste water treatment facility for processing an organic acid becomes unnecessary. Furthermore, according to this hydrogen production method, not only an anaerobic bacterium or facultative anaerobic bacterium having an ability to produce hydrogen using an organic acid as a substrate, but also an anaerobic having an ability to produce hydrogen using a sugar as a substrate. Hydrogen is also produced by culturing sexual or facultative anaerobic bacteria. For this reason, compared to when anaerobic bacteria or facultative anaerobic bacteria having the ability to generate hydrogen using an organic acid as a substrate can be cultured alone, the amount of hydrogen produced can be increased more efficiently. It is possible to produce hydrogen.

  According to the hydrogen production method of the present invention, hydrogen can be produced with high energy conversion efficiency, and the entire equipment can be downsized.

  Hereinafter, preferred embodiments of the present invention will be described in detail.

  The first hydrogen production method of the present invention contains an anaerobic bacterium or facultative anaerobic bacterium (hereinafter referred to as “hydrogen-producing bacterium A”) having an ability to generate hydrogen using an organic acid as a substrate. It is characterized by causing hydrogen producing bacteria A to produce hydrogen by culturing in a medium.

  In such a hydrogen production method, since the organic acid is contained in the culture medium, the hydrogen producing bacterium A produces hydrogen using this organic acid as a substrate. For this reason, it becomes possible to produce | generate hydrogen with high energy conversion efficiency. Further, unlike the case of culturing by light irradiation, a vast culture facility is not required. Furthermore, since an organic acid is not unnecessary, a wastewater treatment facility for treating the organic acid is not required. Therefore, hydrogen can be produced with high energy conversion efficiency, and the entire equipment for carrying out this production method can be reduced in size.

  Here, the organic acid used as a substrate by the hydrogen-producing bacterium A is a carboxylic acid having 1 to 6 carbon atoms because microorganisms are easily metabolized and hydrogen is taken out from the organic acid found in the organic waste liquid. Is preferred. Furthermore, lactic acid, acetic acid, propionic acid, citric acid, succinic acid or malic acid, which are generally present in organic waste at a high frequency and high concentration, are particularly preferred.

  The hydrogen-producing bacterium A exists in various environments, and does not exist in a specific environment. For example, hydrogen producing bacteria A can be found in sludge. More specifically, the hydrogen-producing bacterium A belongs to the genus Clostridium, Citrobacter, Escherichia or Photorhabdus, and has an ability to generate hydrogen using an organic acid as a substrate. The inventors have found that fungi are present. When cultivating the hydrogen-producing bacterium A, it may be cultured in a state contained in the sludge, or the hydrogen-producing bacterium A may be isolated and cultured from the sludge. In order to isolate the hydrogen-producing bacterium A, bacterial groups collected from various environments may be cultured in a medium containing an organic acid and screened using the hydrogen-producing ability as an index. Such screening can be performed by a known method.

  A specific method is, for example, as follows. A medium containing only an organic acid as a carbon source is added to a vial, and a small amount of a sample collected from various environments (for example, sand, mud, seawater, lakes, etc.) is added. After sealing with a butyl stopper, the gas phase of the vial is replaced with an inert gas such as argon. After incubating the vial in an incubator at an appropriate temperature (for example, at 36 ° C. for 24 hours), a part of the gas is collected from the gas phase of the vial and analyzed by gas chromatography. Measure. A sample with a large amount of hydrogen generation is selected, and a part of the culture of the sample is taken out and applied to an agar plate containing the organic acid. The agar plate is cultured using an anaerobic jar under appropriate conditions (eg, 36 ° C., cool and dark place) to form colonies. The appearing colonies are scraped with a platinum wire, added to a vial containing a medium containing only the organic acid as a carbon source, and incubated in the same manner as described above, and then the amount of hydrogen generated is measured. Colonies that generate a large amount of hydrogen are selected and formed on agar plates. By repeating this operation, it is possible to isolate the hydrogen-producing bacterium A.

  In the hydrogen production method of the present invention, it is not necessary to culture the hydrogen-producing bacterium A alone, and it may be cultured together with bacteria that do not have the ability to generate hydrogen using an organic acid as a substrate. Even when culturing together with other bacteria, the hydrogen producing bacterium A can produce hydrogen without significantly inhibiting its ability to produce hydrogen by the other bacteria. Further, a single type of hydrogen producing bacterium A may be cultured, or a plurality of types of hydrogen producing bacterium A may be cultured. In addition, when a plurality of types of hydrogen-producing bacteria A are cultured, various types of organic acids are consumed, so that there is an advantage that the organic acids can be effectively used and the amount of hydrogen generation is increased.

  The medium and culture conditions for cultivating the hydrogen producing bacterium A are not particularly limited as long as the medium and the culture conditions allow anaerobic bacteria or facultative anaerobic bacteria to grow. For example, 37 ° C. (medium temperature fermentation), When the hydrogen producing bacterium A is cultured at a pH of 6.8 to 7.2 and an organic acid concentration of about 3000 ppm, the bacterium can produce hydrogen.

  The above-described culture of hydrogen-producing bacterium A is usually performed by putting hydrogen-producing bacterium A and a medium in a closed culture tank. In this case, the hydrogen-producing bacterium A generates hydrogen using an organic acid contained in the medium as a substrate. The culture is a batch type in which a predetermined amount of hydrogen-producing bacteria A and a medium are charged into a culture tank, and the hydrogen-producing bacteria A generates hydrogen using an organic acid contained in the medium as a substrate. Hydrogen-producing bacteria A ( It is preferable to use a flow method in which a medium is poured into a carrier and the like, and hydrogen is produced by the hydrogen-producing bacterium A using an organic acid contained in the medium as a substrate.

  Examples of a method for taking out hydrogen generated in the culture tank include a method using a hydrogen separation membrane. This is a method that utilizes the properties of hydrogen molecules that are significantly smaller than other gas molecules. Many hydrogen separation membranes are commercially available, and any of them can be used. Specifically, the gas in the culture tank is taken out, the taken-out gas is passed through the hydrogen separation membrane, and the hydrogen that has passed through the hydrogen separation membrane may be recovered in another tank connected to the culture tank.

  The second hydrogen production method of the present invention is characterized in that the hydrogen producing bacterium A is cultured in an organic waste liquid containing an organic acid to cause the hydrogen producing bacterium A to produce hydrogen.

  According to this hydrogen production method, since an organic acid is contained in the organic waste liquid, the hydrogen-producing bacterium A generates hydrogen using this organic acid as a substrate. For this reason, it becomes possible to produce | generate hydrogen with high energy conversion efficiency. Further, unlike the case of culturing by light irradiation, a vast culture facility is not required. Furthermore, since an organic acid is not unnecessary, a wastewater treatment facility for treating the organic acid is not required. In addition, according to the method for producing hydrogen of the present invention, the organic waste liquid that is no longer needed is purified, and at the same time becomes a hydrogen generation source, so that the organic waste liquid can be effectively used.

  In addition to organic residues generated during the manufacturing process of shochu, the above organic waste liquids include waste liquids from food waste (eg, garbage, okara), cans and juice factories, and methane fermentation residue liquids. Etc.

  Of these, organic residues generated during the production process of shochu are preferred. This is due to the following reason. That is, the organic residue generated in the manufacturing process of shochu is generated in Japan over 500,000 tons per year. However, this organic residue contains a large amount of water and is difficult to treat, and most of the organic residue has been dumped into the ocean. On the other hand, this organic residue generally contains a large amount of organic acid. Therefore, by culturing the bacterium according to the present invention in this organic residue, it is possible to process the organic residue that has been difficult to process and to produce hydrogen more efficiently.

  Here, the organic acid contained in the organic waste liquid is preferably a carboxylic acid having 1 to 6 carbon atoms, particularly preferably lactic acid, acetic acid, propionic acid, citric acid, succinic acid or malic acid. . This is because when hydrogen-producing bacteria A are cultured in such an organic waste liquid, it is possible to efficiently generate hydrogen.

  The hydrogen-producing bacterium A may be added to the organic waste liquid as it is, and the culture may be performed. However, in order for the hydrogen-producing bacterium A to efficiently generate hydrogen, the following pretreatment is preferably performed. First, the organic waste liquid is centrifuged to remove suspended solids. Next, a pH adjuster is added to the organic waste liquid from which the suspended solids have been removed to adjust the pH to 6.8 to 7.2. Furthermore, a reducing agent (for example, L-cysteine or ascorbic acid) is added to adjust the redox potential.

  When the hydrogen producing bacteria A and the organic waste liquid are put into the culture tank and the hydrogen producing bacteria are cultured, the hydrogen producing bacteria A generates hydrogen using the organic acid contained in the organic waste liquid as a substrate. As described above, the culture may be either a batch type or a flow type. Further, the method for taking out hydrogen generated in the culture tank is as described above.

  The third hydrogen production method of the present invention comprises an anaerobic bacterium or facultative anaerobic bacterium (hereinafter referred to as “hydrogen-producing bacterium B”) having the ability to produce hydrogen and an organic acid using sugar as a substrate, and hydrogen production. Bacteria A is cultured in a medium containing sugar to cause both hydrogen producing bacteria to produce hydrogen.

  According to this hydrogen production method, when the hydrogen producing bacterium A and the hydrogen producing bacterium B are cultured together, hydrogen and an organic acid are produced using a saccharide contained in the medium as a substrate. Further hydrogen is produced as a substrate. For this reason, it becomes possible to produce | generate hydrogen with high energy conversion efficiency. Further, unlike the case of culturing by light irradiation, a vast culture facility is not required. Furthermore, an organic acid is not unnecessary. That is, even when an organic acid is generated by generating hydrogen using a sugar as a substrate, hydrogen is generated using the organic acid as a substrate and the organic acid is processed. For this reason, the waste water treatment facility for processing an organic acid becomes unnecessary. Furthermore, according to this hydrogen production method, hydrogen is generated not only by culturing the hydrogen producing bacterium A but also by culturing the hydrogen producing bacterium B. For this reason, compared with when hydrogen-producing bacterium A is cultured alone, the amount of hydrogen produced can be further increased, and hydrogen can be produced with higher efficiency.

  The hydrogen-producing bacterium B used in the present hydrogen production method is not particularly limited as long as it is a bacterium that has the ability to produce hydrogen and organic acids using sugar as a substrate. For example, Clostridium spp., Enterobacter spp., Pseudomonas (Pseudomonas) genus bacteria, Bacillus (Bacillus) genus bacteria, etc. are mentioned.

  The culture medium and culture conditions for culturing both hydrogen-producing bacteria are not particularly limited as long as the culture medium and culture conditions allow anaerobic bacteria or facultative anaerobic bacteria to grow. For example, 37 ° C. (medium temperature fermentation), When both hydrogen producing bacteria are cultured at a pH of 6.8 to 7.2 and a sugar concentration of 10 to 30 g / L (in terms of glucose), the bacteria can produce hydrogen.

  When hydrogen-producing bacteria A, hydrogen-producing bacteria B, and a medium are placed in a culture tank and cultured, first, hydrogen-producing bacteria B produce hydrogen using a saccharide contained in the medium as a substrate. At this time, the hydrogen-producing bacterium A generates hydrogen using the organic acid generated simultaneously as a substrate. As described above, the culture may be either a batch type or a flow type. The method for taking out the generated hydrogen is as described above.

  In the fourth method for producing hydrogen of the present invention, the hydrogen-producing bacterium B is cultured in a first medium containing sugar, the hydrogen-producing bacterium B produces hydrogen and an organic acid, and the produced hydrogen-containing bacterium B contains the produced organic acid. The hydrogen producing bacterium A is cultured in the medium 2 to cause the hydrogen producing bacterium A to produce hydrogen. Here, the first medium and the second medium are different.

  According to this hydrogen production method, hydrogen and an organic acid are produced in the first medium by culturing the hydrogen producing bacteria B in the first medium. And the organic acid produced | generated at this time is transferred to a 2nd culture medium. Hydrogen is further generated by the hydrogen-producing bacterium A using the organic acid contained in the second medium as a substrate. For this reason, it becomes possible to produce | generate hydrogen with high energy conversion efficiency. Further, unlike the case of culturing by light irradiation, a vast culture facility is not required. Furthermore, according to this hydrogen production method, an organic acid is not required. That is, even when an organic acid is generated by generating hydrogen using a sugar as a substrate, hydrogen is generated using the organic acid as a substrate and the organic acid is processed. For this reason, the waste water treatment facility for processing an organic acid becomes unnecessary. Furthermore, according to this hydrogen production method, hydrogen is generated not only by culturing the hydrogen producing bacterium A but also by culturing the hydrogen producing bacterium B. For this reason, compared with when hydrogen-producing bacterium A is cultured alone, the amount of hydrogen produced can be increased, and hydrogen can be produced with higher efficiency.

  In addition, if both hydrogen producing bacteria are cultured in the same medium, there is a possibility that the two bacteria may cause an inhibition relationship with each other and culture conditions suitable for producing hydrogen may be different. If both hydrogen producing bacteria are cultured independently in separate culture tanks as in the method for producing hydrogen, hydrogen yield may be increased.

  The medium and culture conditions for culturing the hydrogen-producing bacterium B are not particularly limited as long as the medium and culture conditions allow anaerobic bacteria or facultative anaerobic bacteria to grow. For example, suitable culture of Clostridium spp. The conditions are pH 5.5 to 5.8, culture temperature 25 to 30 ° C., oxidation-reduction potential −150 to −250 mV, and sugar concentration 10 to 30 g / L (in terms of glucose).

  The culture medium and culture conditions for cultivating hydrogen-producing bacterium A are the same as described above, but an organic acid obtained by culturing hydrogen-producing bacterium B is added to the medium.

  In the fourth hydrogen production method, for example, the hydrogen producing bacteria B and the first medium are put in a sealed first culture tank, and the hydrogen producing bacteria A and the second medium are put in a sealed second culture tank. And the organic acid generated in the first medium can be put in the second medium.

  As described above, the culture may be either a batch type or a flow type. In the case of the flow type, it is necessary to install the second culture tank in which the hydrogen producing bacteria A is fixed downstream of the first culture tank in which the hydrogen producing bacteria B is fixed. The method for taking out the generated hydrogen is as described above.

  EXAMPLES Hereinafter, although an Example and a comparative example are given and this invention is demonstrated in more detail, this invention is not limited to these Examples.

  First, the presence of anaerobic bacteria or facultative anaerobic bacteria with the ability to generate hydrogen using organic acids as substrates in samples collected from various environments (sand, mud, seawater, lakes) It investigated in Test Examples 1-6.

(Test Example 1)
Separation and identification of anaerobic bacteria or facultative anaerobic bacteria present in environmental samples having the ability to generate hydrogen using lactic acid as a substrate were carried out by the following method. Yeast extract (3 g), peptone (5 g), ammonium sulfate (1 g), magnesium sulfate heptahydrate (0.2 g), disodium hydrogen phosphate dodecahydrate (14 g), potassium dihydrogen phosphate in 1 L of distilled water (6 g) and iron sulfate heptahydrate (0.0125 g) were added to prepare a solution. To this solution, lactic acid (3 g / L) as a fermentation substrate was added. After adjusting the pH of the solution to 6.8, sterilization (121 ° C., 10 minutes) was performed.

  In a clean bench, the sterilized solution (8 mL) was added to a sample bottle with a volume of 30 mL. Then, after adding a reducing agent to 1% (v / v), an environmental sample was inoculated to 1% (v / v). For the reducing agent, 22% (w / v) L-ascorbic acid, 11% (w / v) sodium carbonate and 2% (w / v) L-cysteine hydrochloride were added to distilled water. A solution obtained by sterilizing by filtration was used.

  After the sample bottle was covered with a rubber stopper and an aluminum cap, the sample bottle was taken out from the clean bench. After replacing the gas phase portion in the sample bottle with argon as an inert gas, the sample bottle was placed in a 37 ° C. constant temperature bath and cultured for 24 hours. Gas was collected from the gas phase portion of the sample bottle, and the collected gas was analyzed by gas chromatography (Shimadzu Corporation, thermal conductivity detector) to quantify the generated hydrogen.

  A sample with a large amount of hydrogen generation was selected, and a part of the sample was applied to an agar plate containing lactic acid and cultured in an anaerobic jar to form colonies. Each obtained colony was cultured in a medium containing lactic acid in the same manner as described above, and the amount of generated hydrogen was quantified. By repeating this operation, anaerobic bacteria or facultative anaerobic bacteria having the ability to generate hydrogen using lactic acid as a substrate were purified and separated.

  The bacteria thus isolated were identified by genetic analysis. That is, the separated bacteria were cultured in a medium containing lactic acid, and a portion thereof was lightly centrifuged to recover the cells. DNA was taken out from the collected bacterial cells, 16S rDNA was amplified by PCR, and the sequence was determined. Based on the determined DNA sequence, a BLAST search was performed with DDBJ, a bacterial species having a high homology was selected, and a bacterial genus having a relevance rate of 96% or more was identified as a separated bacterial genus.

  As a result, Clostridium bacteria (anaerobic bacteria) and Escherichia bacteria (facultative anaerobic bacteria) were isolated and identified as bacteria having the ability to generate hydrogen using lactic acid as a substrate. In addition, when these bacteria were cultured, it was confirmed that succinic acid, formic acid, acetic acid, propionic acid, and butyric acid were produced as fermentation byproducts.

(Test Example 2)
Separation and identification of anaerobic bacteria or facultative anaerobic bacteria present in environmental samples having the ability to generate hydrogen using acetic acid as a substrate were carried out in the same manner as in Test Example 1. As a result, Clostridium spp. And Escherichia spp. Were isolated and identified as those having the ability to generate hydrogen using acetic acid as a substrate. In addition, when these bacteria were cultured, it was confirmed that succinic acid, formic acid, isobutyric acid, and butyric acid were produced as fermentation byproducts.

(Test Example 3)
Separation and identification of anaerobic bacteria or facultative anaerobic bacteria having an ability to generate hydrogen using propionic acid as a substrate in an environmental sample was performed in the same manner as in Test Example 1. As a result, Clostridium spp., Escherichia spp. And Photolabdas spp. (Facultative anaerobic bacteria) were isolated and identified as those having the ability to generate hydrogen using propionic acid as a substrate. In addition, when these bacteria were cultured, it was confirmed that succinic acid, formic acid, acetic acid, and isobutyric acid were produced as fermentation byproducts.

(Test Example 4)
Isolation and identification of anaerobic bacteria or facultative anaerobic bacteria present in environmental samples having the ability to generate hydrogen using citric acid as a substrate were carried out in the same manner as in Test Example 1. As a result, Clostridium spp., Escherichia spp. And Photolabdas spp. Were isolated and identified as fungi capable of generating hydrogen using citric acid as a substrate. In addition, when these bacteria were cultured, it was confirmed that succinic acid, formic acid, acetic acid, propionic acid, isobutyric acid and butyric acid were produced as fermentation byproducts.

(Test Example 5)
Separation and identification of anaerobic bacteria or facultative anaerobic bacteria having an ability to generate hydrogen using succinic acid as a substrate in an environmental sample was performed in the same manner as in Test Example 1. As a result, Clostridium bacteria and Citrobacter bacteria (anaerobic bacteria) were isolated and identified as bacteria having the ability to generate hydrogen using succinic acid as a substrate. In addition, when these bacteria were cultured, it was confirmed that acetic acid and propionic acid were produced as fermentation byproducts.

(Test Example 6)
Isolation and identification of anaerobic bacteria or facultative anaerobic bacteria present in environmental samples having the ability to generate hydrogen using malic acid as a substrate were carried out in the same manner as in Test Example 1. As a result, Clostridium spp., Escherichia spp. And Photolabdas spp. Were isolated and identified as having the ability to generate hydrogen using malic acid as a substrate. In addition, when these bacteria were cultured, it was confirmed that succinic acid, acetic acid, and propionic acid were produced as fermentation byproducts.

(Example 1)
Next, the sample collected from the sludge was subjected to the following experiment as a sample containing anaerobic bacteria or facultative anaerobic bacteria having an ability to generate hydrogen using an organic acid as a substrate. The sludge sample is sludge after sewage sludge is subjected to methane fermentation treatment at a sewage treatment plant and is not pretreated. Using this sludge sample, it was investigated by the following method whether hydrogen could be generated from an organic residue (shochu distiller, hereinafter referred to as “organic residue”) generated during the production process of shochu.

  As an organic residue, a shochu shochu distilled spirit was used. When the composition of this organic acid was analyzed, it was succinic acid: 1237 ppm, lactic acid: 5365 ppm, formic acid: 68 ppm, acetic acid: 1915 ppm, propionic acid: 132 ppm, total sugar amount: 4116 ppm, and glucose: 0 ppm. Distilled rice cake of shochu shochu was obtained from Southern Green Cooperative (Kagoshima Prefecture).

  A sodium hydroxide aqueous solution was added to the organic residue to adjust the pH to 6.8, followed by sterilization (121 ° C., 10 minutes). In a clean bench, sterilized organic residue (8 mL) was added to a sample bottle with a volume of 30 mL. In order to lower the oxidation-reduction potential in the culture solution, a reducing agent was added to the organic residue so as to be 1% (v / v), and then a sludge sample was inoculated so as to be 1% (v / v). . For the reducing agent, 22% (w / v) L-ascorbic acid, 11% (w / v) sodium carbonate and 2% (w / v) L-cysteine hydrochloride were added to distilled water. A solution obtained by sterilizing by filtration was used.

  After the sample bottle was covered with a rubber stopper and an aluminum cap, the sample bottle was taken out from the clean bench. After replacing the gas phase portion in the sample bottle with argon, which is an inert gas, the sample bottle was placed in a constant temperature bath at 37 ° C. and cultured. Gas was collected from the gas phase part of the sample bottle 24 hours and 48 hours after the start of the culture, and the collected gas was analyzed by gas chromatography (Shimadzu Corporation, thermal conductivity detector), and the generated hydrogen and carbon dioxide were quantified. Went. The results are shown in FIG.

  As shown in FIG. 1, when culturing for 24 hours and 48 hours, 3.9 mL and 4.4 mL of hydrogen were generated, respectively. When cultured for 24 hours and 48 hours, 6.1 mL and 7.7 mL of carbon dioxide were generated, respectively, but generation of other gases (such as hydrogen sulfide) was not confirmed.

  From the results of Example 1, hydrogen can be produced by culturing anaerobic bacteria or facultative anaerobic bacteria having the ability to generate hydrogen using an organic acid as a substrate in the organic residue generated in the shochu production process. Was confirmed.

(Examples 2 to 7)
It was investigated by the following method whether hydrogen could be produced from a medium containing an organic acid using a sludge sample.

  First, yeast extract (3 g), peptone (5 g), ammonium sulfate (1 g), magnesium sulfate heptahydrate (0.2 g), disodium hydrogen phosphate dodecahydrate (14 g), diphosphate phosphate in 1 L of distilled water. A solution to which potassium hydrogen (6 g) and iron sulfate heptahydrate (0.0125 g) were added was prepared. Fermentation substrate (3 g / L) was added to this solution, and the resulting solution was used as an organic residue simulation liquid (hereinafter referred to as “simulation liquid”) generated in the shochu production process. As the fermentation substrate, lactic acid, acetic acid, propionic acid, citric acid, succinic acid or malic acid was used (in order, Examples 2 to 7). After adjusting the pH of the simulated solution to 6.8, sterilization (121 ° C., 10 minutes) was performed.

  In a clean bench, a sterilized simulated solution (8 mL) was added to a sample bottle with a volume of 30 mL. Then, the same reducing agent as in Example 1 was added to 1% (v / v), and then a sludge sample was inoculated to 1% (v / v).

  After the sample bottle was covered with a rubber stopper and an aluminum cap, the sample bottle was taken out from the clean bench. After replacing the gas phase portion in the sample bottle with argon as an inert gas, the sample bottle was placed in a 37 ° C. constant temperature bath and cultured for 24 hours. Gas was collected from the gas phase portion of the sample bottle, and the collected gas was analyzed in the same manner as in Example 1 to quantify the generated hydrogen. The results are shown in FIG.

  FIG. 2 is a graph showing the amount of generated hydrogen when various organic acids are cultured as fermentation substrates. Table 1 shows the maximum amount of hydrogen generated when each organic acid is used.

  From the results of Examples 2 to 7, it is confirmed that hydrogen can be produced by culturing anaerobic bacteria or facultative anaerobic bacteria having the ability to generate hydrogen using an organic acid as a substrate in a medium containing an organic acid. It was done.

It is a graph showing the hydrogen generation amount at the time of culture | cultivating in an organic residue. It is a graph showing the amount of hydrogen generation when various organic acids are cultured as a fermentation substrate.

Claims (6)

  An anaerobic bacterium or facultative anaerobic bacterium having an ability to produce hydrogen using an organic acid as a substrate is cultured in a medium containing an organic acid, and the bacterium produces hydrogen. .   The method for producing hydrogen according to claim 1, wherein the organic acid is composed of at least one selected from the group consisting of lactic acid, acetic acid, propionic acid, citric acid, succinic acid, and malic acid.   An anaerobic bacterium or facultative anaerobic bacterium having an ability to generate hydrogen using an organic acid as a substrate is cultured in an organic waste liquid containing an organic acid, and the bacterium generates hydrogen. Production method.   The method for producing hydrogen according to claim 3, wherein the organic waste liquid is an organic residue generated during the production process of shochu.   An anaerobic bacterium or facultative anaerobic bacterium having the ability to generate hydrogen and an organic acid using sugar as a substrate, and an anaerobic bacterium or facultative anaerobic bacterium having an ability to generate hydrogen using an organic acid as a substrate. A method for producing hydrogen, comprising culturing in a medium containing bacterium to cause both bacteria to produce hydrogen. An anaerobic bacterium or facultative anaerobic bacterium having the ability to generate hydrogen and organic acid using sugar as a substrate is cultured in a first medium containing sugar, and the bacterium generates hydrogen and organic acid. An anaerobic bacterium or facultative anaerobic bacterium having an ability to generate hydrogen using an organic acid as a substrate in a second medium containing the organic acid, and causing the bacterium to generate hydrogen. , Hydrogen production method.

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