JP3549444B2 - Method for producing hydrogen by microorganisms - Google Patents

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for producing hydrogen by a microorganism. More specifically, the present invention relates to a method for efficiently producing hydrogen using solar energy by using microalgae having photosynthetic ability and bacteria having photosynthetic ability.
[0002]
[Prior art]
In recent years, global warming has been regarded as a major cause of global unusual weather and disrupting the harmony of the global ecosystem, and prevention of global warming has become an urgent issue worldwide. This global warming is caused mainly by the massive consumption of fossil fuels, mainly oil and coal, and the accompanying carbon dioxide (CO2). ₂ It is clear that greenhouse gases are the cause, and the search for new technologies to control global warming gas emissions, creation of energy sources such as hydrogen using biological functions, fixation and decomposition of greenhouse gases, etc. Research has become necessary.
[0003]
Among them, hydrogen energy can be converted to electric energy with high efficiency as a fuel cell, the calorific value is 3 to 4 times that of petroleum, water is generated after combustion, and there is no risk of environmental pollution. It has certain advantages.
[0004]
Various researches on the creation of hydrogen energy using biological functions have been carried out. For example, JP-A-58-60992 discloses that a green algae having photosynthetic ability and hydrogen-producing ability is cultured under light and aerobic conditions. A method for decomposing a substance accumulated in photosynthesis under dark aerobic conditions to generate hydrogen is disclosed. Also, Pharmacia: vol. 26, 419-422 (1990), describes that among the green algae, the MGA161 strain of the genus Chlamydomonas has a high hydrogen-producing ability. Further, Biosci. Biotech. Biochem. : Vol. 56, 751-754 (1992) discloses that the photosynthetic product of the Chlamydomonas sp. Is described as being generated efficiently. In this document, it is described that hydrogen production by photosynthetic bacteria is performed using ethanol, acetic acid, formic acid, lactic acid, and the like, which are fermentation degradation products of a green algal photosynthetic product, as electron donors. Describes that hydrogen production is not carried out, and it is not clear by what mechanism hydrogen is produced, and thus the conditions for optimal hydrogen production have not yet been established.
[0005]
On the other hand, the reason why the method for producing hydrogen by microorganisms is not performed on an industrial scale is a problem of a culture device. For example, culture of photosynthetic green algae and photosynthetic bacteria requires light, but the light cannot reach the deep part of the culture tank. Therefore, it is necessary to make the culture tank shallow, and therefore, there is a locational problem that the floor area must be increased for mass cultivation, and because the culture tank is shallow, dissolution of carbon dioxide in the culture solution is difficult. There was a problem in culture conditions that the culture was insufficient and the culture was inefficient. Also, continuous culture was difficult.
[0006]
In order to solve these problems, a method of culturing with stirring by applying artificial light was also studied.However, there was a problem in that the light did not reach the inside sufficiently, the irradiation time of light to the cells, and the irradiation amount, In addition, the problem of mass production of hydrogen has not been solved due to the cost of artificial light and the like.
[0007]
[Problems to be solved by the invention]
Therefore, in a system for producing hydrogen using microalgae and photosynthetic bacteria, efficient cultivation of photosynthetic microalgae and establishment of optimal hydrogen production conditions for photosynthetic bacteria using fermentation degradation products of photosynthetic products as substrates are expected. I was
[0008]
[Means for Solving the Problems]
The present invention has been made in order to solve the above-mentioned problems, and it is an object of the present invention to efficiently culture photosynthetic microalgae and optimal hydrogen production conditions for photosynthetic bacteria using a fermentation degradation product of a photosynthetic product as a substrate and hydrogen production by microorganisms. It provides a mass production method.
[0009]
The present invention provides a step of culturing microalgae having photosynthetic ability under light and aerobic conditions to obtain a photosynthetic product, a step of culturing the microalgae under dark anaerobic conditions to obtain a fermented liquid of the photosynthetic product, and the fermented liquid A method for producing hydrogen comprising a step of allowing a bacterium having photosynthetic ability to act under light and anaerobic conditions, wherein the microalga is cultured under the light and aerobic conditions and / or the bacteria are allowed to act on the fermented liquid under light and anaerobic conditions. The present invention relates to a method for producing hydrogen, wherein the step of performing is performed in a transparent tower type air lift culture tank.
[0010]
In a preferred embodiment, the tower-type air lift culture tank has a double cylindrical structure.
[0011]
In a preferred embodiment, the tower-type air lift culture tank has a structure that generates a helical flow.
[0012]
In a further preferred embodiment, a step of culturing the microalgae having photosynthetic ability under light and aerobic conditions to obtain a photosynthetic product, and a step of culturing the microalgae under dark anaerobic conditions to obtain a fermented liquid of the photosynthetic product And a step of allowing bacteria having photosynthetic ability to act on the fermentation broth under light and anaerobic conditions.
[0013]
In a preferred embodiment, the step of allowing bacteria having photosynthetic ability to act on the fermentation broth under light-anaerobic conditions is performed by adjusting the lactic acid content to at least 0.3 mM.
[0014]
The present invention also provides a step of culturing microalgae having photosynthetic ability under light and aerobic conditions to obtain a photosynthetic product, a step of culturing the microalgae under dark anaerobic conditions to obtain a fermented solution of the photosynthetic product, and A method for producing hydrogen comprising a step of allowing bacteria having photosynthetic ability to act on a fermentation liquor under bright anaerobic conditions, wherein the lactic acid content of the fermentation liquor under the dark anaerobic conditions is adjusted to at least 0.3 mM. And how to.
[0015]
BEST MODE FOR CARRYING OUT THE INVENTION
As the microalgae used in the present invention, any microalgae having photosynthetic ability can be used without particular limitation. In particular, green algae, cyanobacteria and the like are preferably used. The green algae, Chlamydomonas reinhardtii (Chlamydomonas) Chlamydomonas line Hull di belonging to the genus (Chlamydomonas reinhardtii), Chlamydomonas Moebushii (Chlamydomonas moewusii), MGA161 shares of Chlamydomonas genus Chlamydomonas Yugametasu (Chlamydomonas eugametos), Chlamydomonas Segunisu (Chlamydomonas segnis), chlorella ( Chlorella vulgaris belonging to the genus Chlorella, Senedesmus obliguus belonging to the genus Sendesmus and Dunaliella belonging to the genus Dunaliella And Dunaliella tertrolecta. Examples of cyanobacteria include Anabaena variabilis ATCC 29413 belonging to the genus Anabaena, and Cyanothece sp. Belonging to the genus Cyanothece. ATCC 51142, Synechococcus sp. Belonging to the genus Synechococcus. PCC 7942 and Anacystis nidulans belonging to the genus Anacystis.
[0016]
Among them, the green alga Chlamydomonas MGA161 strain is a strain isolated from seawater by a conventional method, and has high photosynthetic ability under light-aerobic conditions, fast fermentation under dark anaerobic conditions, and light-aerobic conditions. When returned to the lower side, it has an excellent ability to quickly adapt to the photosynthesis system, and is therefore preferable. The Chlamydomonas MGA161 strain or a strain corresponding thereto is a strain that can be easily isolated by a person skilled in the art from a seawater sample with photosynthetic ability and resolution.
[0017]
Culture of microalgae under bright and aerobic conditions is carried out in a medium containing inorganic components under irradiation of sunlight and / or artificial light using a transparent tower-type airlift culture tank. The culturing temperature is about 15 to 40 ° C, preferably 25 to 35 ° C.
[0018]
The medium for culturing microalgae, particularly green algae, is not limited as long as it is a medium containing an appropriate nitrogen supply source and an inorganic component. Seawater is preferably used as a medium containing an inorganic component, but a modified Okamoto medium (hereinafter referred to as a MOM medium) having the following composition:
NaCl 30 g
CaCl ₂ ・ 2H ₂ O 200 mg
MgSO ₄ ・ 7H ₂ O 250 mg
FeSO ₄ ・ 7H ₂ O 20 mg
KH ₂ PO ₄ 40.8mg
K ₂ HPO ₄ 495 mg
Vitamin B ₁ 100 μg
Vitamin B ₁₂ 1 μg
1M NH ₄ Cl 5 ml
Trace metal mixture A5 1.0 ml
Distilled water 1000 ml pH 8.0
Is also used.
Here, the composition of the trace metal mixture A5 is as follows.
H ₃ BO ₄ 2.85 g
MnCl ₂ ・ 4H ₂ O 1.81 g
ZnSO ₄ ・ 7H ₂ O 0.22 g
CuSO ₄ ・ 5H ₂ O 0.08 g
Na ₂ MoO ₄ 0.021g
CaCl ₂ ・ 6H ₂ O 0.01 g
EDTA · 2Na 50 g
Distilled water 1000 ml
[0019]
By the way, hydrogen production by photosynthetic bacteria under light-anaerobic conditions generally depends on nitrogenase. This nitrogenase activity is subject to inhibition depending on the concentration of the nitrogen source (eg, ammonium chloride, glutamic acid). In the present invention, a photosynthetic product may be fermented and decomposed under dark anaerobic conditions by using a bright aerobic culture solution as it is or by concentrating microalgae, and this may be used for hydrogen production under light anaerobic conditions. Therefore, the nitrogen source concentration of the medium under bright and aerobic conditions is preferably as low as possible. In the case of ammonium chloride, the concentration is adjusted to 5 mM or less, preferably 2.5 mM or less, more preferably 0.5 mM or less. In the case of other nitrogen sources, the concentration is preferably as low as possible so as not to inhibit nitrogenase.
[0020]
Therefore, even in the cultivation of microalgae, especially green algae, the MOM medium (NH ₄ (Cl concentration: 5 mM), and is preferably adjusted to the lowest possible ammonium chloride concentration, preferably 0.5 mM or less. By adjusting the ammonium chloride concentration to 0.5 mM or less, the starch accumulation concentration can be made higher than in the case of 5 mM.
[0021]
In the present invention, since carbon dioxide is required as a carbon source for culturing microalgae, air or a mixed gas of air and carbon dioxide is aerated. When mixing carbon dioxide, about 2 to 10%, preferably about 2 to 5% carbon dioxide is mixed.
[0022]
In the present invention, since sunlight is used using a transparent tower-type air-lift culture tank, light irradiation is not particularly necessary.However, in consideration of insufficient sunshine hours such as cloudy weather and rainy weather, light irradiation is performed. Equipment may be provided. In addition, when culturing continuously at night, it is necessary to provide light irradiation equipment.
[0023]
The material of the transparent tower type air lift culture tank may be any material as long as it is transparent and can transmit sunlight and / or artificial light. For example, (reinforced) acrylic resin, transparent plastics such as polycarbonate, polypropylene, and polyterephthalate, and glass are exemplified.
[0024]
In addition, a heating and / or cooling device can be provided at the upper and / or lower part of the culture tank to adjust the temperature of the culture solution.
[0025]
The form of the air lift culture tank is not limited as long as it has a ventilation device such as a nozzle, a ring-shaped sparger, or a perforated plate, and can circulate the liquid by this. The light (sunlight or artificial light) applied to the airlift culture tank hits the cells (microalgae) in the vicinity of the tank wall of the culture tank best. However, as the amount of cells increases, it hardly reaches the inside, so photosynthesis occurs. (Proliferation) becomes insufficient. Also, too much light will adversely affect cell growth. Therefore, appropriate fluidity of cells and appropriate mixing of cells inside the tank and cells outside (surface side) of the tank are required.
[0026]
In the present invention, it is preferable that the inside of the transparent tower type air lift culture tank has a double cylindrical structure. Hereinafter, description will be given based on FIG. The culture tank 1 used in the present invention is preferably cylindrical, and an inner cylinder 2 (hereinafter, may be simply referred to as a “cylinder”) may be provided at the lower part of the culture tank 1 with a space enough to allow a liquid to flow. It is set up so that the culture solution 3 inside the cylinder 2 and the culture solution 4 outside the cylinder 2 circulate. By aerating from a lower portion inside the cylinder 2 provided in the culture tank through a ventilation device 5 (for example, a ring-shaped sparger), a flow of the culture solution from the lower portion to the upper portion inside the cylinder 2 is formed. The culture solution is made to flow from the upper surface inside the cylinder 2 to the outside of the cylinder 2. Then, the culture solution flows to the lower portion of the culture tank along the space between the tank wall 6 and the cylinder 2 to create a circulation state of the culture solution. The culture solution is strongest while flowing between the tank wall 6 and the cylindrical outer wall 7, and is irradiated with light (sunlight) so that photosynthesis proceeds. As described above, by forming the inside of the transparent tower type air lift culture tank 1 into a double cylindrical structure, the microalga culture solution flows between the tank wall 6 and the cylindrical outer wall 7, and the light irradiation time on the microalga is reduced. Since the microalgae can be lengthened and the microalgae are efficiently circulated, the photosynthesis (proliferation) of the microalgae is performed more efficiently than in the case of using a conventional stirring type culture tank.
[0027]
The cylinder used in the present invention is also preferably transparent. The material may be the same as that of the transparent culture tank, or may be a different material.
[0028]
In the present invention, it is preferable that the tower type air lift culture tank has a structure that generates a helical flow. The helical flow is described in Merchuk et al. (3RD LINENAL CONFERENCE ON Bioreactor and Bioprocess Fluid Dynamics, pp. 61-68). Generally, in a conventional circulation type air lift culture tank, the flow in the tank is only upstream and downstream, cells near the outer surface receive strong light, growth inhibition occurs, and cells inside receive light required for photosynthesis. I can't. Therefore, a radial flow is applied so that the light energy required for photosynthesis is evenly applied to all cells. For this purpose, for example, a device (helical flow promoter) 8 for generating a helical flow can be used. As shown in FIG. 1, this device 8 is provided with several fins or baffles 9 so as to generate a spiral or radial flow when rotated in water. By attaching to the upper part of the inner cylinder 2 of the tank 1, the culture solution flows out radially or spirally toward the downstream. When this device is arranged above the inner cylinder 2, agitation occurs while flowing between the tank wall 6 and the cylindrical outer wall 7, so that the microalgae are not exposed to excessive light (sunlight) and cultured. The liquid is irradiated with light uniformly and for a long time, and photosynthesis proceeds more efficiently than when the liquid is simply made into a double cylinder structure.
[0029]
The size of the airlift culture tank and the size of the inner cylinder of the present invention may be appropriately determined in consideration of the material and the culture scale.
[0030]
In the present invention, the aeration rate may be determined in consideration of the culture rate. In general, when a helical flow device is used, cells can be fluidized even if the aeration amount is smaller than when no helical flow device is used. If the air flow rate is too large, air pockets are generated in the helical flow device, which is not preferable.
[0031]
In this microalga culture step, it is desirable to collect microalgae in the logarithmic growth phase as much as possible. This is because fermentation (decomposition of photosynthetic products) under the following dark anaerobic conditions is performed smoothly. Further, it is preferable to culture the microalga by continuous culture because the microalga has a high growth rate and can maintain a logarithmic growth phase.
[0032]
The obtained microalgae is transferred to a fermenter, and a step of fermenting the photosynthetic product under dark anaerobic conditions is performed. In this step, a photosynthetic product such as starch is decomposed and fermented, and organic acids such as formic acid, acetic acid and lactic acid, and alcohols such as ethanol and glycerol are released outside the microalgae. These organic substances are used as electron donors for hydrogen production by photosynthetic bacteria. Accordingly, the dark anaerobic condition is preferably a condition under which so-called fermentative production of organic matter is smoothly performed, and particularly preferably a condition under which a large amount of lactic acid is fermented.
[0033]
The fermenter used for fermentation under dark anaerobic conditions is not particularly limited. It is preferable to make the upper gas phase space of the fermenter as small as possible. In order to maintain anaerobic conditions, carbon dioxide or a mixture of carbon dioxide and inert gas is introduced into the upper space to create an atmosphere with little or no oxygen to promote the decomposition of photosynthesis products, It is preferable to suppress the occurrence of as much as possible. Bubbling of an inert gas is not preferred. The stirring may be very gentle.
[0034]
Fermentation of microalgae under dark anaerobic conditions is performed by increasing the concentration of microalgae from the viewpoint of increasing the concentration of organic acids or alcohols in the decomposition solution that is used as a substrate for photosynthetic bacteria under the following bright anaerobic conditions. Is preferred. Therefore, it is preferable to concentrate the microalga culture solution under bright and aerobic conditions using a concentration / separation means such as membrane separation and centrifugation. In the case of continuously culturing under bright and aerobic conditions, microalgae are transferred to a fermenter under dark and anaerobic conditions while being continuously concentrated.
[0035]
The fermentation temperature of the microalga under dark anaerobic conditions is about 10 ° C to 40 ° C, preferably about 25 ° C to 35 ° C.
[0036]
In the fermentation of microalgae under dark anaerobic conditions, it is preferable that lactic acid is secreted into the decomposition solution. In the present invention, it was shown that lactic acid induces the production of nitrogenase. That is, hydrogen is generated only when lactic acid is present. The lactic acid concentration is preferably at least 0.3 mM or more.
[0037]
In addition, in the step of obtaining a fermentation solution under dark anaerobic conditions, a relatively low dilution ratio, that is, by performing continuous fermentation with a long residence time, to continuously obtain a fermentation solution having a high degree of decomposition of photosynthetic products. This is preferable because it can be continuously supplied to the next hydrogen generation step under light-anaerobic conditions.
[0038]
The decomposed liquid (fermented liquid) obtained by fermenting microalgae under dark anaerobic conditions is separated from the microalgae and used as a substrate for hydrogen production by photosynthetic bacteria under the following light anaerobic conditions. All or part of the microalgae is again used for photosynthesis under bright aerobic conditions.
[0039]
The same culture tank as that used for culturing the microalgae under the bright and aerobic conditions is used as the transparent tower-type air lift culture tank used in the step of allowing photosynthetic bacteria to act under light and anaerobic conditions.
[0040]
The anaerobic condition is created by passing an inert gas such as argon, hydrogen, or a mixed gas of carbon dioxide and hydrogen. From the viewpoint of recovering the generated gas, hydrogen or a 1: 2 mixed gas of carbon dioxide and hydrogen is preferable. It is preferable to circulate hydrogen while extracting part of it. The culture temperature of the photosynthetic bacterium is about 10C to 40C, preferably about 25C to 35C.
[0041]
As the photosynthetic bacterium, photosynthetic vegetative bacterium and photosynthetic organotrophic bacterium (such as red sulfur-free bacterium and green gliding bacterium) are used. In the present invention, since an organic substance generated by decomposing a photosynthetic product is used as a substrate (electron donor), a photosynthetic organotrophic bacterium is preferably used. Examples of the photosynthetic organotrophic bacterium include a red sulfur-free bacterium belonging to the family Rhodospirillaceae, a green gliding bacterium belonging to the family Chloflexaceae, and the like.
[0042]
Such a photosynthetic organotrophic bacterium can be selected, for example, by selecting a microorganism that generates hydrogen from a seawater sample using lactic acid (eg, 0.3 mM) as a substrate. The photosynthetic bacterium, for example, Rhodopseudomonas (Rhodopseudomonas) belonging to the genus Rhodopseudomonas Parasutorisu (Rhodopseudomonas palustris) and Rhodopseudomonas Ashidofira (Rhodopseudomonas acidophila), Rodosupiriramu rubrum (Rhodospirillum rubrum) ATCC 11170 belonging to Rodosupiriramu (Rhodospirillum) genus, the IFO 3986 Etc., belonging to the genus Rhodobacter, Rhodobacter spheroides, Rhodobacter capsulatus ATCC 23782, ATCC 1 7013, etc., belonging to the genus Rhodovulum, Rhodovulum strictum, Rhodovulum adriaticum, Rhodovulum sulfilum, and the like.
In the present invention, a strain named Rhodobram sulphidophilum W-1S (hereinafter sometimes simply referred to as W-1S) isolated from a seawater sample and a photosynthetic bacterium having an activity equivalent thereto are preferably used. Can be
[0043]
The photosynthetic bacteria under light-anaerobic conditions are preferably immobilized on a carrier. This is because it is recyclable, does not particularly require growth, and is preferable for continuously treating the fermented liquid.
[0044]
For immobilization of photosynthetic bacteria, for example, known methods such as a carrier binding method, a cross-linking method and an entrapping method can be applied, but the carrier binding method is most suitable. The carrier binding method includes a chemical adsorption method or a physical adsorption method in which an ion-exchange resin is adsorbed. As the material of the carrier used in the present invention, for example, foams or resins such as porous glass beads, polyvinyl alcohol, polyurethane foam, polystyrene foam, polyacrylamide, polyvinyl formal resin porous body, silicon foam, cellulose porous body, etc. Is preferred. The size of the opening of the porous body is preferably about 10 μm to 500 μm.
[0045]
The shape of the carrier is not limited, but in consideration of the strength of the carrier, culture efficiency, etc., the shape is spherical or cubic, and the size is 2 mm to 50 mm in the case of a sphere, and 2 mm to 50 mm in the case of a cubic. Is preferred.
[0046]
Further, as described above, nitrogenase involved in hydrogen production of photosynthetic bacteria is inhibited by nitrogen, so that the nitrogen source concentration in the reaction solution under light-anaerobic conditions is preferably as low as possible.
[0047]
In addition, the fermented broth under dark anaerobic conditions contains organic acids (formic acid,
Acetic acid, lactic acid, etc.) and alcohols (ethyl alcohol, glycerol, etc.), which serve as electron donors to generate hydrogen. Among them, as shown in Example 1, it was found by the present inventors that lactate was an inducer of a nitrogenase involved in the generation of hydrogen, and it was proportional to the lactate concentration (about 0.3 mM). As a result, it was confirmed that nitrogenase was induced. Therefore, it is preferable to keep the lactic acid concentration at least at least 0.3 mM in order to promote the generation of hydrogen. Therefore, a mode in which hydrogen is generated under bright and anaerobic conditions while adding lactic acid is also included in the scope of the present invention.
[0048]
Further, since organic acids other than lactic acid, for example, malic acid and succinic acid can also induce nitrogenase, malic acid and succinic acid can be used instead of lactic acid, or may be used in combination with lactic acid.
[0049]
Further, the microalgae are continuously cultured under bright and aerobic conditions (that is, light is irradiated at night), and the obtained microalgal alga bodies are sequentially supplied to fermentation under dark anaerobic conditions. By continuously supplying the fermentation broth under the dark anaerobic condition to the photosynthetic bacterium under the light anaerobic condition, continuous culture and hydrogen production become possible.
[0050]
【Example】
Hereinafter, the present invention will be described with reference to examples using green algae as microalgae, but the present invention is not limited to these examples.
(Example 1)
(Culture of green algae using transparent tower type airlift culture tank)
FIG. 1 shows a culture tank 1 used under the bright aerobic and bright anaerobic conditions used in the present invention. An acrylic resin cylinder 2 having an inner diameter of 17.5 cm and a height of 346 cm is arranged at a position 4 cm from the bottom of a cylindrical acrylic resin culture tank 1 having an inner diameter of 28 cm and a height of 380 cm (about 230 L in volume). In addition, a double cylindrical structure is used in which the culture solution in the culture tank 1 can circulate between the inside of the cylinder and the outside (the space formed by the culture tank wall 6 and the outer surface 7 of the cylinder). A ring-shaped sparger 5 having a diameter of 12 cm was placed 10 cm from the bottom inside the cylinder. This sparger was a tube having a diameter of 1 cm, and had 50 1 mm holes. A helical flow promoter 8 was arranged at the upper part of the cylinder, and a helical flow was generated in the space between the inner wall 6 of the culture tank and the outer wall 7 of the inner cylinder from the upper part to the lower part of the culture tank.
[0051]
The bright and aerobic conditions are sunlight and light source (18W / m ² ). As the medium, a modified Okamoto medium was further modified, and ammonium chloride was used by dissolving it to a concentration of 0.5 mM. The green alga Chlamydomonas MGA161 strain was inoculated in a 200 L medium at a rate of 3.5 μg dry cell weight / ml, and air containing 5% carbon dioxide was ventilated at 0.3 vvm to start the culture, and the logarithmic growth phase was started. The continuous culture was started when The dilution rate was 0.083 / hr.
[0052]
The growth rate of the green alga MGA161 strain in this culture was 0.267 g / L / hr. This value indicates that the same volume of culture solution was used at 18 W / m ² This was about twice the value obtained when the cells were cultured in a 35 cm deep raceway-type culture tank (actual volume: 200 L) while aerating with 0.3 vvm of air containing 5% carbon dioxide. Further, the floor area of the transparent air lift type culture tank used in this experiment was about 1/10 of the raceway type culture tank. Therefore, the production volume of algal cells per unit floor area of the transparent air lift type culture tank was about 20 times that of the raceway type culture tank, indicating the effectiveness of the transparent air lift type culture tank.
[0053]
(Decomposition of photosynthesis products and hydrogen production under light and anaerobic conditions)
The culture solution extracted in the continuous culture was placed in a small continuous membrane separator, and transferred to a fermenter under dark anaerobic conditions while concentrating the concentration of green algae about twice. This fermenter had a capacity of 100 L made of stainless steel and had one stirring blade. The fermentation under dark anaerobic conditions was performed at about 30 ° C., and was performed with very gentle stirring while circulating carbon dioxide in the gas phase.
[0054]
Fermentation under dark anaerobic conditions was carried out for 12 hours, and after completion of the fermentation, the green algae and the fermentation broth were separated. On the other hand, the lactic acid concentration of the fermentation broth was 0.32 mM. The lactic acid concentration was determined by separation by HPLC (high performance liquid chromatogram) and quantitative analysis by absorbance at 210 nm.
[0055]
The photosynthetic bacterium used in the light and anaerobic condition is W-1S, and sodium succinate 1.0 g / l, sodium acetate 1.0 g / l, sodium malate 1.0 g / l, and sodium pyruvate 1.0 g in advance. / L, aerobically cultured in a modified Okamoto medium containing 1 mM ammonium chloride at 30 ° C for 24 hours. This photosynthetic bacterium (W-1S) was added so as to have a dry cell amount of 2 g per liter of the fermentation broth. Culture under light anaerobic conditions was carried out at 30 ° C. while aerating and circulating a mixed gas of carbon dioxide and hydrogen at a ratio of 1: 2 at 1 vvm. The generated hydrogen was separated and recovered from carbon dioxide by a method of passing through an alkaline solution.
[0056]
The culture tank used was of the same type as that used for culturing the green algae of Example 1, and the amount of fermentation solution to be added and the position of the helical flow promoter were also the same.
[0057]
As a result of the reaction for 12 hours, hydrogen was generated in an amount of 585 ml / g dry cells. This amount of generated hydrogen was equivalent to about 2.6 times the amount of hydrogen generated under the same conditions using a parallel plate photobioreactor having a height of 50 cm and the same volume. The floor area of the transparent air-lift type culture tank was about 1 / 7.5 of that of the parallel plate type photobioreactor. Therefore, the amount of hydrogen generated per unit floor area of the transparent air lift type culture tank was about 19 times that of the parallel plate type photobioreactor, indicating the effectiveness of the transparent air lift type culture tank.
[0058]
(Example 2)
The effects of organic acids on hydrogen production by photosynthetic bacteria under light and anaerobic conditions were investigated. In a modified Okamoto medium containing 0.6 mM acetic acid, 1.5 mM ethanol and 0.5 mM glycerol, lactic acid was added to each of 0, 0.1, 0.2, 0.3, 0.4, 0.5 and After adding 1 mM, the amount of generated hydrogen of W-1S was measured. FIG. 2 shows the results. In FIG. 2, no hydrogen was generated in the medium without lactic acid, whereas hydrogen was generated in the medium with lactic acid. From these results, it was found that lactic acid induced production of nitrogenase. Hydrogen evolution increased in a concentration-dependent manner up to 0.3 mM, but not further. Therefore, it was found that if at least 0.3 mM or more of lactic acid was contained, the amount of generated hydrogen was almost constant.
[0059]
Although not described as data, it was revealed that malic acid and succinic acid also induce production of nitrogenase.
[0060]
【The invention's effect】
In the production of hydrogen using microorganisms, the efficient cultivation of microalgae under bright aerobic conditions and / or the production of hydrogen by photosynthetic bacteria under bright and anaerobic conditions by using a transparent tower type air-lift culture tank. Can be.
[Brief description of the drawings]
FIG. 1 is a schematic view of a culture tank used in the present invention.
FIG. 2 is a diagram showing that lactic acid is essential for hydrogen production and that the optimal lactic acid concentration is 0.3 mM or more.
[Explanation of symbols]
1 Culture tank
2 Internal cylinder
3. Culture solution inside the inner cylinder
4. Culture solution outside the inner cylinder
5 Ventilation device
6 Tank wall of culture tank
7 Outer wall of inner cylinder
8 Helical flow promoter
9 Baffles

Claims (4)

Culturing the microalgae having photosynthetic ability under light and aerobic conditions to obtain a photosynthetic product, culturing the microalgae under dark anaerobic conditions to obtain a fermented solution of the photosynthetic product, and In a method for producing hydrogen comprising a step of allowing a bacterium having photosynthetic ability to act under conditions, the step of culturing the microalgae under the aerobic condition and / or the step of causing the bacterium to act on the fermented solution under the anaerobic condition is transparent. A method for producing hydrogen, which is performed in a tower-type airlift culture tank, wherein the tower-type airlift culture tank has a double cylindrical structure and has a structure that generates a helical flow. The method according to claim 1, wherein the bacteria having photosynthetic ability is immobilized on a carrier. Culturing the microalgae having photosynthetic ability under light and aerobic conditions to obtain a photosynthetic product; culturing the microalgae under dark anaerobic conditions to obtain a fermented solution of the photosynthetic product; and The method according to claim 1 or 2, wherein the step of causing the bacteria having photosynthetic ability to act under anaerobic conditions is performed continuously. The method according to any one of claims 1 to 3, wherein the lactic acid content of the fermentation liquor under the light-anaerobic condition is adjusted to at least 0.3 mM.

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