JPH08308591A - Biological production of hydrogen - Google Patents

Abstract

PURPOSE: To provide an innovative biological hydrogen production method by an entirely new conception enabling stable mass production of hydrogen. CONSTITUTION: In this method for biologically producing hydrogen from an organic substrate, yeast and obligatory anaerobic, heterotrophic and hydrogen- productive Clostridium bacteria are mixedly cultured under anaerobic conditions in the organic: substrate at pH5.5-6.5 under an oxidation-reduction potential, of -150 to -250mV. The organic substrate is e.g. organic wastewater, organic waste including sludge.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for biologically producing hydrogen from an organic substrate, and in particular, a mixed culture of bacteria and yeast using organic waste such as sludge and waste water as a substrate. Regarding hydrogen production method.

[0002]

2. Description of the Related Art Recently, global environmental problems have rapidly become apparent on a global level, and in particular, there is concern about global warming due to carbon dioxide derived from fossil fuels. For this reason, research and development of microbiological hydrogen gas production technology that does not depend on fossil fuels and does not cause environmental pollution has been vigorously carried out.
Microorganisms that produce hydrogen gas are roughly classified into photosynthetic microorganisms and non-photosynthetic microorganisms. As photosynthetic microorganisms that produce hydrogen gas, photosynthetic microorganisms (microalgae) that decompose water to generate oxygen under irradiation of light energy include the following genus species.

The other is a photosynthetic bacterium that releases excess hydrogen as a gas in the process of assimilating organic matter,
The main genera include the following genera and species. ＊ Non-red sulfur bacterium ・ Rhodospirillum genus ・ Rhodopseudomonas ＊ Red sulfur bacterium ・ Chromatium genus ・ Thiocapsa genus Research on hydrogen production and / or organic wastewater treatment by photosynthetic microorganisms is the earth. 19 before environmental problems became apparent
Since 1975, he has been energetically conducting research results that should be evaluated. Stable continuous hydrogen production by polymer gel immobilization technology of photosynthetic microorganisms using a certain strain of marine photosynthetic bacterium Chromatium or marine cyanobacterium Oscillatoria The achievements were the treatment of wastewater (concentration of concentrated wastewater is not possible) and hydrogen production, or the technology of hydrogen production and nitrogen removal by synchronized dark and light culture, and the results of these researches were "Hydrogen production by photosynthetic microorganisms" (edited by Shuichi Suzuki). Biomass / Energy Conversion, Kodansha Scientific, pp. 194-228 [198]
3]).

However, despite the fact that this enormous and valuable research result has been obtained in the process from the past to the present, there is a hydrogen production facility or a wastewater treatment facility which is currently operating on a practical scale. do not do. It is thought that the main reasons are as follows. Continuous irradiation of solar energy is essential for the growth of photosynthetic microorganisms. Cultivation of photosynthetic bacteria usually requires a vast area. Deactivation of nitrogenase, which is a hydrogen reductase, immobilization technology for stabilizing hydrogenase, etc. are necessary, and technical difficulties are involved. A dense culture technique and a culture device to reduce the required area are required. (Because of cost increase) Concentration of sunlight, even distribution of light in the culture tank (enhancement of energy use efficiency).

[0005] In contrast to the hydrogen gas production by the photosynthetic bacteria, research on hydrogen production by non-photosynthetic bacteria has been extensively conducted. This kind of hydrogen-producing bacterium belongs to obligate or facultative anaerobic bacterium, and its representative bacterium is as described above, but it decomposes organic matter in the process of heterotrophic growth and occurs in this process. Hydrogen is produced by reducing excess electrons with hydrogenase, and the reducing power associated with energy production is automatically adjusted by hydrogen production. This anaerobic heterotrophic hydrogen-producing bacterium is capable of decomposing organic matter and producing hydrogen as a result, so that high-concentration organic waste and other biomass can be recycled as energy and treated and disposed of at the same time. Although it is possible and can be a potential environmental protection technology, hydrogen production by this heterotrophic hydrogen-producing bacterium has the following drawbacks at this stage.

A fermentation reaction formula for hydrogen production from an organic substance (represented by glucose) by an anaerobic bacterium is usually represented by Formula 1. C ₆ H ₁₂ O ₆ + 2H ₂ O = 2CH ₃ COOH + 2CO ₂ + 4H ₂ Formula 1 (standard free energy = + 184 kj / mol) As can be easily understood from Formula 1, hydrogen produced from 1 mol of glucose. Is only 4 moles, which is an extremely small amount relative to 12 moles of hydrogen-producing bacteria of photosynthetic bacteria (formula 2). _{C 6 H 12 O 6 + 6H} 2 O = 6CO 2 + 12H 2 ········· formula 2 biggest cause of the hydrogen production disparities, the hydrogen production by photosynthetic bacteria, the driving force of the reaction Adenosine triphosphate (ATP) is theoretically infinite as long as at least solar energy is supplied, whereas hydrogen production from organic matter by anaerobic bacteria is
184 to finally break down organics into water and carbon dioxide
The energy of kj / mol is insufficient. Therefore, hydrogen fermentation by anaerobic bacteria is a complete absorption ergon reaction, and various lower fatty acids accumulate in the fermentation liquor in an amount corresponding to the insufficient ATP amount. Therefore, not only the hydrogen production amount is small but also the fermentation digestion Secondary treatment of the liquid is required.

Insufficient ATP amount = 184 kj / mol / 30.5 kj / mol.ATP.apprxeq.6.0 mol.ATP / mol.C ₆ H ₁₂ O ₆ ... Formula 3 ATP by anaerobic bacteria (hydrogen-producing bacteria) The amount of production is 7 mol, which is 4 mol per mol of glucose under the ideal conditions in the glycolysis system and 3 mol in the acid fermentation process, and unless the ATP is supplemented or supplemented by some means or method, the hydrogen fermentation is more than this. It does not proceed in the positive direction, and reaches a dynamic equilibrium state when lower fatty acids are accumulated. In order to solve this problem, the following studies are known (hydrogen fermentation; hydrogen production from biomass, microorganisms, Vol. 3, No. 6, pp. 42-4).
9, 1987). That is, anaerobic bacteria do not need light irradiation, have a strong ability to decompose organic substances, and can produce hydrogen from biomass. Further, there is an advantage that the culture can be performed in a large tank without the need for ventilation. However, the drawback is that organic substances cannot be completely decomposed and organic acids are accumulated,
The hydrogen generation rate is also low.

On the other hand, photosynthetic bacteria generally have low assimilation performance for organic substances, but they can utilize light energy to completely decompose organic substances into water and carbon dioxide. Also, products of anaerobic fermentation such as organic acids are preferably used. As described above, since anaerobic bacteria and photosynthetic bacteria have a complementary relationship in the metabolic function of the substrate, it is considered that the substrate can be completely decomposed by using these two types of bacteria in combination. Based on this idea, Clostridium butyricum (Clos
It has been reported that, when tridium butyricum) and Rhodopseudomonas sphaeroides were mixed and cultured, 1 to 7 mol of glucose could be recovered. On the other hand, when the fermentation effluent of Clostridium butyricum was fed to the photosynthetic bacteria instead of the mixed culture, only 4 mol or less of hydrogen was obtained in total. It is reported that a synergistic effect that increases the generation efficiency is also considered to occur. However, the hydrogen production amount in the mixed culture based on this idea is at most 7 mol,
There is still a considerable difference when compared to 12 moles of photosynthetic bacteria.

Further, before that, an experiment of hydrogen production by mixed culture of anaerobic bacteria and photosynthetic bacteria was conducted, and the following results were obtained. In other words, glucose (+ artificial medium containing various necessary components) was used as a substrate, and Clostridium butyricum was used as an anaerobic hydrogen-producing bacterium.
tyricum) IFO 13949, a newly discovered non-sulfur photosynthetic bacterium Rhodopse
udomonas) SP ・ RV (Rhodopseudomonas capsulata ・
... deposited as FERM P-7254),
At a culturing temperature of 30 ° C., and under anaerobic conditions, 10 klux of light irradiation (anaerobic conditions) was performed for mixed culture, and 7 mol to 9 mol of hydrogen was calculated from 1 mol of glucose (J. Ferment.Technol., Vol. 62, No. 6, p.
531 to 535, 1984, JP-B-63-49994). In any case, when hydrogen is produced from biomass such as organic waste by interposing photosynthetic microorganisms, artificial light energy use cannot be expected from the viewpoint of hydrogen production cost. When relying on sunlight for energy, it is practically impossible to provide permanently stable light energy.

[0010]

DISCLOSURE OF THE INVENTION As described in detail above,
There is a strong demand for the emergence of water treatment technology and organic waste recycling technology that can achieve hydrogen production as well as the processing of biomass such as organic waste, but currently practical technologies have been developed. It has not been. An object of the present invention is to solve the above-mentioned problems of the prior art and to provide an innovative biological hydrogen production method based on a completely new idea that can stably produce a large amount of hydrogen.

[0011]

In order to solve the above-mentioned problems, the present invention provides a method for biologically producing hydrogen from an organic substrate, wherein the organic substrate is an obligately anaerobic heterotrophic substance. A biological hydrogen production method is characterized in that a bacterial group of the genus Clostridium that has the ability to produce hydrogen and yeast is mixed and cultured under anaerobic conditions. In the hydrogen production method of the present invention, the mixed culture has a pH of 5.5.
~ 6.5, especially near pH 6, redox potential of -150 ~
It is good to carry out at -250 mV, especially near -200 mV,
As the organic substrate, organic wastewater and organic waste including sludge can be used.

As described above, according to the present invention, an obligately anaerobic heterotrophic hydrogen-producing bacterium uses various organic wastes as substrates to produce hydrogen in a glycolytic system and a TCA cycle system, and is simultaneously produced. Yeasts for brewing lower fatty acids that belong to fungi,
For example, the genus Saccharomyces and / or
Or wild yeasts such as Candida genus, Pichia genus, Debaryomyces
Yeasts belonging to the genus Hansenula and the like are mixed and co-cultured under obligate anaerobic conditions, and ATP, which is a high-energy substance produced in this anaerobic environment, is hydrogenated by an obligate anaerobic heterotrophic hydrogen-producing bacterium. It is a system for producing a large amount of hydrogen from organic waste by supplying it to a production reaction and coupling the biological reactions of both fungi with ATP.

In the method of the present invention, the obligate anaerobic heterotrophic wild-habited in the organic waste rich in organic matter such as ordinary soil, various concentrated organic wastewater and / or various sludges. Focusing on the hydrogen-producing bacteria, by effectively utilizing the function of the bacteria, organic substances such as fiber (cellulose), carbohydrates, fats, proteins and / or lower fatty acids, which are the main constituents of organic waste, are decomposed. The first feature is that it is decomposed and made into low molecular weight compounds in the sugar system and TCA cycle system. The surplus electrons generated in the microbial cells during this decomposition process become hydrogen by hydrogenase and are carried out of the microbial cells along with water (hydrogen production), and the reducing power and the reducing atmosphere accompanying energy production are adjusted to the reduced form of NAD (NADH). ) Is adjusted by generating

In the case of ordinary microorganisms, electrons are added to oxygen by the current reaction inside the mitochondria, that is, by electron transfer by cytochromes to generate water, and in combination with it, the driving force for microbial activity and function expression. ATP is produced in an appropriate amount, but the genus Clostridium of the obligately anaerobic hydrogen-producing bacterium of the present invention has NAD → FAD → UQ as an electron transfer system, but lacks the cytochrome electron transfer system of liver kidney. are doing. Therefore, the bacterial group of the genus Clostridium has basically a small amount of ATP production in the glycolytic system and the TCA cycle system.
The hydrogen fermentation reaction becomes an absorption ergon reaction, and energy is insufficient to achieve a complete reaction. Therefore, there is a fatal defect that a large amount of lower fatty acids are accumulated in the fermentation digestion liquid, and the hydrogen production amount is only about 30% of the photosynthetic microorganism. The hydrogen fermentation reaction formulas when this Clostridium genus and the photosynthetic bacterium introduced as a conventional technique use glucose as a substrate are shown in the above Formulas 1 and 2, but in the hydrogen fermentation by the Clostridium genus, the reaction achievement rate is 1
To achieve 00%, the standard energy is 184 kj /
Mol-glucose is deficient and this calorie is roughly equivalent to 6.0 mol ATP / mol glucose.

It is also considered that the genus Clostridium, which is the main feature of the present invention, may be replaced by the genus Entobacter (Klebsiera) which is a facultative anaerobic heterotrophic hydrogen-producing bacterium widely distributed in nature. However, both of these strains have deficiencies in the electron transfer system, and the free energies obtained from the ergon reaction, which is the energy generation reaction (decomposition / catabolism) in the previous step, are coupled to consume the energy. The absolute amount of ATP, which plays a central role in transferring to the reaction (hydrogen generation reaction) process, is insufficient, and the production of the target substance, that is, the hydrogen production amount is significantly limited. When a facultative anaerobic bacterium is used for hydrogen production, it inhibits the enzymatic stability of hydrogenase, which functions as an outlet for surplus electrons.
Nitrogenase activity is irreversibly inhibited by oxygen contamination and mixing.

What should be considered as an important factor is that various organic wastes, which are the target substrates for hydrogen production in the present invention, are one of the main characters of the present invention when left in a natural environment. It is not possible that only Clostridium bacteria alone constitute an ecosystem, and various anaerobic and aerobic microorganisms coexist or coexist,
This is the fact that it constitutes a so-called mixed culture system. An important bacterium which is directly related to the present invention and whose presence cannot be ignored is methane bacterium, and depending on the culture conditions of the heterotrophic hydrogen-producing bacterium, methane bacterium becomes the dominant species in the mixed culture system, However, there is a risk that the intended hydrogen production will be hindered. However, it has been experimentally verified and confirmed that there is a considerable disparity between the optimal conditions required for living and growing in the natural environment between Clostridium and methane bacteria. By controlling the above, the genus Clostridium, which is the object of the present invention, can be always proliferated as a dominant species in the mixed culture system.

The controlling factors are as follows. * There is a considerable difference in growth rate between Clostridium and Clostridium. * There is a considerable difference in the optimum pH range between the two genus species, with Clostridium having a pH of 5.5 to 5.8, whereas methane bacteria have a pH of around 7.8. * There is a considerable disparity between the two genus species in the preferred redox potential (ORP) as a fermentation environment. -Clostridium genus -150 to -250 mV-Methane bacterium -350 to -450 mV * Both genus species differ in sensitivity to hydrogen, and Clostridium is quite sensitive to methane bacteria and is active due to the presence of hydrogen in the fermentation environment. Easy to be hindered.

As described above, since there is a considerable difference in susceptibility to the above environmental factors between the genus Clostridium and the methane bacterium, as described above, also in the present invention, the genus Clostridium is always the dominant species and hydrogen fermentation is performed. , P of mixed cultures to ensure hydrogen production without delay
In order to adjust H to around 5.5 and redox potential to around −200 mV by artificial means and operations, and further to eliminate the poisoning effect of hydrogen, and to improve the adsorption ergon reaction of hydrogen fermentation, as necessary. It is preferable to carry out vacuum fermentation or slightly anaerobic hydrogen fermentation. A second feature of the present invention is that in a method of biologically producing hydrogen from an obligate anaerobic heterotrophic hydrogen-producing bacterium, Clostridium, using organic waste as a fermentation substrate, the ATP-producing ability of the bacterium is insufficient. Complement,
In order to complete the hydrogen fermentation reaction of the bacterium, a specific brewing yeast and / or wild yeast is mixed and co-cultivated at a constant ratio,
Using the ATP produced by the yeasts in the anaerobic fermentation process, various lower fatty acids remaining in the fermentation digestion liquid are converted into hydrogen, and the photosynthetic microorganisms produce the total hydrogen production amount under the light energy supply condition (anaerobic condition). It is to provide a method for surely obtaining a hydrogen production amount comparable to 12 mol / mol-glucose achievable under bright conditions).

The reaction formulas for completing hydrogen fermentation by supplying ATP from the genus Clostridium and yeasts in the features 1 and 2 of the present invention are shown below. * ATP supplied from the hydrogen production _{C 6 H 12 O 6 + 2H} 2 O = 2CH 3 COOH + 4H 2 + 2CO 2 ···· formula 4 * brewer's yeast and / or wild yeast by clostridium
2CH ₃ COOH + 6H ₂ O = 8H ₂ + 4CO ₂ ··· Formula 5 * Hydrogen production in a symbiotic system of Clostridium and yeast C ₆ H ₁₂ O ₆ + 6H ₂ O = 12H ₂ + 6CO ₂ ... Formula 6 H ₂ / C ₆ H ₁₂ O ₆ = 12 mol / mol

Usually, the substance synthesis reaction in the living body of a microorganism is
Almost all of the energy (7.7 kcal / mol · ATP) when ATP or the like is hydrolyzed is used in conjugation with the substance synthesis. If such an energy coupling reaction becomes industrially available, it becomes possible to make the synthetic reaction in the microbial organism, which is an absorption ergon reaction, proceed in the forward direction. It is considered that if can be conjugated, it can proceed in the positive direction. The problem with this technology is that since ATP, which is an energy source in a bioreactor for synthesizing substances, is expensive, adding commercially available ATP to the reaction system aims to produce substances with high added value. It is not practical unless it is a biological reaction, and there is little possibility of further practical application by the microbiological hydrogen production method.

Therefore, in order to apply this basic idea, in addition to the enzyme system that governs the substance synthesis reaction, ATP is used as energy in the bioreactor for synthesis and AMP produced as a result of reduction is used. An enzyme system is required to reproduce ADP. Another technical problem is that even if ATP is reproduced at low cost,
It is whether or not this ATP is used not only for enzymatic reaction but also for microbial reaction by live bacteria without any resistance. Even if the ATP reproduction system and the microbial reaction system are coupled, the substrate used for the ATP reproduction system is expensive,
It is difficult to apply to environment-related technologies, such as strong stability of the enzyme used. It is known that yeasts, especially baker's yeast and alcohol-fermenting yeast, have an ATP production amount several times higher than that of ordinary bacteria.

Study on ATP reproduction using baker's yeast (Yeast bioscience, pp.141-15)
4, Academic Society Publishing Center, 1990), by utilizing the strong energy released in the glycolysis process of yeast fermentation, using glucose and azinosine as substrates and supplying phosphoric acid, ATP 50 to 100 mg / ml from baker's yeast. Is producing. However, AT obtained by biological reaction by baker's yeast
The reactions coupled with P are all combinatorial reactions with enzymes of different biological origins, and the possibility of substance production by coupling the regenerated ATP with the microbial reaction has not been confirmed.
As mentioned above, A is considered to be a promising technical means for improving the ergonomic reaction of hydrogen fermentation by Clostridium sp.
We investigated the possibility of an energy (ATP) -conjugated bioreactor that incorporates a TP reproduction system and a microbial synthesis reaction or a decomposition reaction, but the ATP reproduction system is costly and the biological hydrogen production method is not It turned out that there is no possibility of application.

As a result of earnest studies on a method for fundamentally solving this technical problem, the present inventors have found that the yeasts described above for the bacterial group of the obligate anaerobic heterotrophic hydrogen-producing bacterium Clostridium, namely, Brewing yeast, for example, Saccharomyces and / or wild yeasts such as Candida, Pichia, Debaryomyces, Hansenula, etc. experimentally confirmed that yeasts belonging to the genus are suitable as symbiotic microorganisms, the yeasts produced By co-cultivating the bacterium as it is with an anaerobic hydrogen-producing bacterium, ATP was supplied without delay to the bio-reaction of ergon of hydrogen production, and it was possible to stably produce a large amount of hydrogen. The reason why the present inventor has selected brewing yeasts and / or wild yeasts as a partner to be mixed and co-cultivated with a bacterial group belonging to the obligate anaerobic hydrogen-producing bacterium Clostridium is described below.

(1) Yeasts belonging to fungi include ATP in the glycolytic pathway regardless of whether they are brewing yeasts or wild yeasts.
The production is strong and reliable, and commercially available ATP is usually industrially produced from yeasts. The theoretical formula of alcohol fermentation when glucose is used as a substrate is as follows. C ₆ H ₁₂ O ₆ → 2C ₂ H ₅ OH + 2CO ₂・ ・ ・ Equation 7 Alcohol fermentation reaction is recognized by many microorganisms, but among them, yeasts are the most powerful and glycolytic The amount of ATP produced per mol of glucose by the series of reactions in the route is 4 mol. 2 mol of this is used for sugar phosphorylation, while the remaining 2 mol is surely used for other microbial reactions. Also, the free energy generated from all reactions in alcohol fermentation is said to be about 52 kcal, but this amount of energy is about 7 mo of ATP.
It corresponds to the amount of heat energy of l. As described above, the amount of ATP produced by yeasts can sufficiently supplement the amount of energy insufficient for the obligate anaerobic hydrogen-producing bacterium Clostridium to complete the hydrogen fermentation reaction.

(2) Yeasts have an innate characteristic of directly supplying ATP produced by the glycolysis system of organic substances to the sucking ergon reaction of other microorganisms. If this property is effectively used, by culturing Clostridium and yeasts having a strong ATP-producing ability in a mixed symbiotic (conjugate) culture, ATP lacking in hydrogen fermentation is complemented, and the hydrogen fermentation reaction is converted into an ergon reaction. Can be converted. (3) Most yeasts have an optimum pH around pH 5.0.
There is a range, while bacteria belonging to the genus Clostridium also have optimum values at pH 5.0-6.0. Therefore, an environment for mixed symbiotic culture of both genera is easy to construct. (4) The redox potential suitable for the yeasts to live and grow is -100 to -2 due to the constitution of the enzyme system and electron transfer system.
00 mV is preferable, but the obligate anaerobic hydrogen-producing bacterium Clostridium, which forms a symbiotic system with these yeasts, prefers a living environment of about the same redox potential, and artificially adjusts to this redox potential. By controlling, the mixed culture system can always be the dominant species over methane bacteria. (5) Some alcohol produced by yeasts is easily decomposed by Clostridium and other bacteria, and as a result, ATP is additionally produced by various anaerobic wild bacteria, which is an ergonomic reaction. Promotes the forward progress of the hydrogen fermentation reaction.

(6) As a practical problem, the biggest concern of Japanese beer factories is the effective use of surplus yeast generated from the manufacturing process. Currently, a very small amount of surplus yeast is effectively used. However, most of them are treated and disposed of as organic waste. The surplus yeast discharged from the factories (about 40 factories) of the four major beer companies in Japan is 135,0.
It is a huge amount of up to 00 tons / year (370 tons / day), and if an effective utilization method can be established, it will be a very large market. Therefore, if this surplus yeast is used properly, it becomes possible to simultaneously treat both as a yeast inoculating hydrogen production from organic waste, and hydrogen can be produced at the same time as the treatment of organic waste. In addition, by using waste yeast wastewater from a beer manufacturing plant as a substrate and inoculating this with bacteria of the genus Clostridium, construct a self-contained factory or system capable of producing hydrogen at the same time as treating waste yeast wastewater. You can Since there are many other alcohol manufacturing plants in Japan, the biological hydrogen production method of the present invention can also be applied to hydrogen production from alcohol distillation wastewater discharged from these plants.

The above is the main reason for selecting brewing yeasts and / or wild brewing as partners in order to complete the reaction of the obligately anaerobic heterotrophic hydrogen-producing bacterium. In the mixed co-cultivation of the genus Clostridium of the present invention with brewing yeast and / or wild yeast under anaerobic conditions, reliable data are available on the glycolytic pathway and the amount of ATP produced by the TCA cycle (acid fermentation step). Although not available, Table 1 shows the ATP production amount (glucose substrate) and the ATP consumption amount at each stage, including assumptions and assumptions. Table 1 shows hydrogen production from organic wastes by anaerobic mixed co-cultivation of Clostridium and yeasts such as Saccharomyces and Candida, and ATP balance in biological reactions at each stage.

[0028]

[Table 1]

Although there are some assumptions and estimates for ATP production and consumption, from the results of ATP balance calculation in Table 1,
The progress of the hydrogen fermentation reaction due to the symbiosis of Clostridium and yeasts is predicted as follows from the viewpoint of ATP production and consumption. (1) In hydrogen fermentation with Clostridium, 4 ATP is produced in glycolysis system and 3 ATP is produced in TCA cycle system. In this system, AT is used for lactic acid fermentation not related to hydrogen generation.
Since P is expected to be partially consumed, the substantial ATP production is estimated to be around 6 ATP. (2) AT in hydrogen fermentation when glucose is used as a substrate
P consumption is 6AT if calculated from the amount of free energy
The hydrogen fermentation and production of P, and thus Clostridium alone, cease at this stage. (3) ATP by brewing yeasts and / or wild yeasts
The amount of production is 2 mol, and the amount of heat generated by alcohol fermentation is 52 kcal / mol C ₆ H ₁₂ O ₆ in the standard state, so surplus ATP that can be supplied to other adsorption ergon reaction.
Is estimated to be (2 + α) mol.

(4) The amount of ATP produced by wild anaerobic bacteria other than the above-mentioned two genus species in the mixed culture system was about 2 mol.
It is estimated that some ATP is required to complete hydrogen fermentation by the obligate anaerobic heterotrophic hydrogen-producing bacterium Clostridium and to secure a hydrogen amount of 12 mol / mol C ₆ H ₁₂ O ₆ equivalent to that of a photosynthetic microorganism. There will be an excess. A for lactic acid fermentation
Assuming that TP is not consumed at all (1.8 + α) m
Assuming that ol is consumed, (0.8 + α) mol of ATP is in excess. Therefore, if the genus Clostridium and the yeast for brewing and / or the wild yeast are mixed and co-cultivated as in the present invention, complete decomposition of the usable organic matter contained in the organic waste and the theoretical amount of hydrogen are produced. be able to.

The obligately anaerobic heterotrophic hydrogen-producing bacterium Clostridium, which plays a major role in the biological hydrogen production method of the present invention, is widely distributed wildly in nature, and in particular, it treats urban sewage and general excreted urine. It lives extremely universally in so-called organic waste such as various organic sludge discharged from the facility or concentrated organic wastewater. The heterotrophic hydrogen-producing bacteria that live in these organic wastes are not only obligate anaerobic bacteria, but also hydrogen-producing bacteria that belong to the category of facultative anaerobic bacteria, and the main bacteria are Citrobacter. (Ci
The heterotrophic hydrogen-producing bacterium, which belongs to the genus trobacter, the genus Entrobacter, etc., and which normally lives in the organic waste as a mixed culture system, is an obligate anaerobic heterotrophic hydrogen-producing bacterium Clostridium. The genus is overwhelmingly
Coexisting with other heterotrophic hydrogen-producing bacteria, it decomposes organic matter as an electron donor, and releases excess hydrogen in the electron transfer system outside the body.

However, as described above, in the genus Clostridium, since the amount of ATP produced in the catabolism reaction (energy production) in the preceding stage of hydrogen fermentation in all steps is small, the hydrogen production reaction (assimilation reaction) becomes an adsorption ergon reaction and the organic matter. The hydrogen production reaction stops while a part of the above remains in the fermentation digestive liquid in the form of lower fatty acid (see Formula 1). Therefore, the BOD of the fermented digestive juice is considerably high, and usually secondary treatment by biooxidation is indispensable. In carrying out the present invention, a specific strain of the obligately anaerobic heterotrophic hydrogen-producing bacterium Clostridium should be selected from, for example, strains registered in ATCC, and this should be treated by increasing culture by a conventional method. Although it may be inoculated to the organic waste, it is more effective to use the mixed culture system of the genus Clostridium that naturally inhabits the organic waste to be treated as it is. A naturally-occurring mixed culture system is expected to have a mutual physiological and functional synergistic effect between coexisting bacterial species, and is preferable to the use of a single pure microorganism as an inoculum.

The present inventor shows a case of carrying out hydrogen fermentation of human waste. The mixed culture system after continuously culturing Clostridium spp.
It was confirmed to be composed of multiple obligately anaerobic heterotrophic hydrogen-producing bacteria. Clostridium butyricum ATCC 25779 * Clostridium barkeri ATCC 25849 * Clostridium cellulolyticum * Clostridium cellulolyticum ATCC 35319 * Clostridium dyspolicum * Clostridium disporicum ATCC 43838 * clotritrium ATCC 43838 * Clostridium AT Closium When organic waste is used as a substrate for hydrogen fermentation, hydrogen-producing bacteria useful for organic waste can be obtained even if a specific pure strain is not purchased from a microorganism preservation institution such as ATCC and inoculated into the hydrogen fermentation reactor. It naturally inhabits, and hydrogen fermentation is performed without resistance due to the synergistic action of these.

If the conditions necessary for hydrogen fermentation are set, the growth of methane bacteria is suppressed, the hydrogen-producing bacteria group most suitable for the input composite substrate is naturally constructed, and the hydrogen-producing bacteria are artificially inoculated. It was confirmed and verified that more stable hydrogen fermentation is performed. Hydrogen fermentation by the obligately anaerobic heterotrophic hydrogen-producing bacterium Clostridium is an ergonomic reaction, and the ATP produced in the catabolism reaction is insufficient. It has already been mentioned in the description that it does not proceed. The technical center of the present invention is to break this dynamic equilibrium state by biological means and to complete the uncompleted hydrogen fermentation by supplying ATP produced by yeasts. This is an innovative technology whose main idea is to produce the same amount of hydrogen (see Formula 2). For yeasts that play this important role, it is desirable and preferred to use brewing yeasts and / or wild yeasts which are widely distributed in nature.

The yeast used in the fermented alcoholic beverage is
Although there are characteristics and differences depending on the type of alcoholic beverage, the following types of yeast are used. * Saccharomyces * Schizosaccharomyces * Saccharomycodes * Saccharomycodes * Pichia * Torulopsis * Candida * Rhodotorula Alcohols listed above Fermentative yeast may be purchased as a pure strain from a microorganism preservation institution such as ATCC, and cultured in a conventional manner in an expanded culture to inoculate a hydrogen fermentation substrate of the genus Clostridium, but as described above, in Japan, it is extremely large from a beer manufacturing plant. Excessive yeast or waste yeast wastewater is discharged, and there is a demand for effective utilization of the wastewater and establishment of appropriate treatment technology.

As the brewing yeast that is symbiotic with the genus Clostridium in the present invention, it is preferable to use the beer surplus yeast and / or the brewer yeast that is present in large amounts in the waste yeast wastewater, rather than using a pure strain. . The so-called brewer's yeasts currently used for beer production in Japan are as follows, and each company is making use of the latest biotechnology technology to create a new type of yeast (variant). * Upper surface yeast Saccharomyces cerevisiae * Lower surface yeast Saccharomyces ca rlsbergensis Saccharomyces uvarum As mentioned above, the brewer's yeast currently in use has the strongest alcohol fermentation ability and is of the genus Saccharomyces. ,
It is optimal as a brewing yeast for supplying ATP of the present invention, and by using the surplus yeast of beer production for this purpose, the surplus yeast can be effectively used, or the purpose of treatment / disposal can be achieved at the same time. You can

Wild yeasts are widely inhabited in nature. The functions of these wild yeasts are not limited to alcohol fermentation, but they catabolize and assimilate polysaccharides, monosaccharides, proteins, oils and fats, and other organic substances to decompose or produce various substances. The main wild yeasts are, for example, Candida, Pichia, Hansenula, Debarilomyces, etc., but all of these yeasts surely produce ATP in the catabolism reaction, so substances synthesized in the assimilation reaction In any case, it can be used for the purpose of the present invention, and its effect can be evaluated to be equivalent to that of yeast for brewing. These wild yeasts are used in the Clostridium spp.
When used for the purpose of complementing TP, for example, ATC
Obtain a specific strain from a microorganism preservation institution such as C.
ATP was obtained by culturing in an expanded amount using the medium defined in C and inoculating a hydrogen fermenter of the genus Clostridium with an appropriate amount.
The purpose of supply is achieved.

As described above, according to the present invention, hydrogen fermentation by the obligate anaerobic hydrogen-producing bacterium Clostridium causes an ergon reaction due to a shortage of ATP production amount. Therefore, since the complete decomposition of organic matter is not completed, the hydrogen production amount is small. In addition, an innovative biological hydrogen production method was invented with the purpose of fundamentally solving the fatal defect that secondary treatment is indispensable for the accumulation of large amounts of lower fatty acids in fermentation digestive juice. is there. In order to drastically improve this technical problem, in the present invention, an obligate anaerobic hydrogen-producing bacterium Clostridium and a brewing yeast and / or a wild yeast are mixed and co-cultivated under anaerobic conditions, and only Clostridium is cultivated. In terms of production amount, complement the amount of ATP, which is insufficient to complete the hydrogen fermentation reaction, with the production amount of yeasts, complete the biological reaction described above, and almost completely decompose the organic matter that is a pollutant source and simultaneously produce a large amount of hydrogen. It is an innovative energy production method based on a new idea and idea.

[0039]

EXAMPLES The present invention will now be described in detail with reference to examples, but the present invention is not limited to these examples. Example 1 Hydrogen production from beer factory wastewater by mixed symbiotic culture of obligate anaerobic heterotrophic hydrogen-producing bacterium Clostridium spp. And yeast Saccharomyces spp.
It is wort squeezing wastewater and waste yeast wastewater. Waste yeast wastewater is liquid-side wastewater used for beer production, in which so-called brewer's yeast is roughly separated into liquid and solid matter by gravity, and naturally contains a considerable amount of brewer's yeast. Therefore, in this verification test, wort squeezing wastewater and waste yeast wastewater discharged from a certain beer factory were mixed at a water ratio of 5: 1, and this mixed wastewater was used as an estimated concentration in a hydrogen fermentation experiment. Table 2
Shows the analytical values of the individual wastewaters and the calculated values of the mixed wastewater quality obtained by mixing the both at 5: 1.

[0040]

[Table 2] (Note) Unit is mg / liter except pH. The quality of mixed wastewater is calculated.

The wastewater specifically used for the hydrogen fermentation experiment was prepared by mixing both wastewaters shown in Table 2 into a 10-liter plastic tank (assuming a storage tank) and storing it in a refrigerator at 5 ° C. for the duration of the experiment. After both were mixed, a precipitation treatment was carried out for a certain period of time to remove some precipitated solid matter. Table 3 shows the water quality analysis results of the mixed wastewater actually used for hydrogen fermentation.

[Table 3]

First, 900 ml of the mixed wastewater shown in Table 3 is placed in a semi-continuous anaerobic fermentation tank having a volume of 2 liters (effective volume of 1 liter) and a constant temperature water tank of 30 ° C. On the other hand, the obligate anaerobic heterotrophic hydrogen-producing bacterium belonging to the genus Clostridium is from ATCC to Clostridium butyricum ATCC859.
And inoculate the liquid medium of the medium ATCC38,
Cultures were expanded at ℃. This Clostridium butyricum
100 ml of an ATCC 859 expanded culture was inoculated into the above anaerobic fermenter, and the total amount was adjusted to 1 liter to perform acclimatization culture. Initially, the mixed wastewater shown in Table 3 was supplied to the fermentation tank in semi-continuous manner from ⅕ of the final load at intervals of 4 days, and the load was gradually increased so that the total load would be reached after about 50 days. . The operating conditions of the anaerobic fermentation tank at this point are roughly as follows.

Operating conditions of anaerobic hydrogen fermenter * Number of days of culture: 10 days, * Culturing temperature: 30 ° C, * Average BOD load: 2.6 to 3.3 kg / m ³ /
Day, * Average CODcr load: 3.5-4.5 kg / m ³ /
* The initial pH in the hydrogen fermentor was around 5.3, but the pH after reaching the dynamic equilibrium state was in the range of 5.8 to 6.3, so pH adjustment was especially necessary. There wasn't. * Since the redox potential (ORP) in the hydrogen fermentor was -175 mV in the steady state, no artificial control was performed.

The acclimatization was completed after about 50 days under the above operating conditions, and the gas generation and the water quality of the fermented digestion solution also showed stable values. Therefore, about 60 days after the start of operation, about 20 days, every other day. The amount of hydrogen generated was accurately measured, and the total amount of sugar (in terms of glucose) and the total amount of sugar removed from the fermentation digestive liquid were calculated to determine the amount of hydrogen generated as mol · H ₂ / mol · C ₆ H ₁₂ O.
Calculated as ₆ . The result is shown in FIG. In parallel with this experiment, as a control experiment, a hydrogen fermentation experiment was carried out on a mixed wastewater that was not inoculated with the expanded culture solution of Clostridium butyricum ATCC859, but virtually no hydrogen fermentation was observed. Was not specified. In FIG. 1,-●-indicates the amount of hydrogen generated, and-○-indicates the pH value.

Experimental Results (1) With the mixed wastewater alone in the beer manufacturing plant, the pH of the mixed culture solution was 7.5 to 7.8 and the ORP was 360 to 410 mV. In the anaerobic fermentation tank, methane fermentation was prioritized and hydrogen gas was not generated. It was only a trace. (2) Heterotrophic hydrogen-producing bacterium Clostridium butyricum ATCC859 is inoculated into a mixed wastewater containing brewer's yeast and anaerobic fermentation is carried out, hydrogen fermentation takes priority, and about 60
Hydrogen was stably generated when the day passed, and the removal rates of total BOD and total CODcr in this process were not shown in the figure, but were 95-98% of the total during the experiment period of about 20 days. The high rate was maintained.

(3) The amount of hydrogen generated in the mixed co-cultivation of the mixed wastewater containing the heterotrophic hydrogen-producing bacterium Clostridium and the yeast for brewing is about 10 mol · H ₂ / mol · C ₆ H
_It showed a high value near ₁₂ O ₆ . In addition, the yeast Saccharomyces genus for brewing was densely detected in the mixed wastewater, the fermentation digestion liquid, and the excess sludge. (4) From the results of the above-described demonstration experiment, by the mixed co-cultivation of the mixed hydrogen waste water (the presence of Saccharomyces genus), which is the biological hydrogen production method of the present invention,
ATP in the process of yeast production is coupled with the hydrogen-producing reaction of the genus Clostridium, and the absorption ergon reaction is converted to the ergon reaction, so that the hydrogen fermentation reaction is almost completed, and hydrogen that is close to the theoretical amount can be produced reliably. Was proved. In addition,
Although we have not particularly conducted experiments on surplus brewer's yeast, it is certain that similar results will be obtained.

Example 2 Clostridium genus of obligate anaerobic heterotrophic hydrogen-producing bacterium and wild yeast mesh Nicowire (Me
Hydrogen production from the first settling tank sludge of municipal sewage by mixed co-cultivation of the genus tschnikowia) The first settling tank sludge (raw sludge) of a standard sewage treatment plant constructed in a certain city of H prefecture in Japan As a test sample, the method of the present invention and the amount of hydrogen produced by the genus Clostridium alone were compared and examined. Sediment (removal by slanting method) and coarse solids were removed from the collected first settling tank sludge in advance, and stored in a large refrigerator at 5 ° C. The daily usage amount was taken out each time, and an effective volume of 1 liter was anaerobic. It was supplied to a digestion tank (semi-continuous culture tank ... 2 liters in volume). Table 4 shows the water quality analysis values (period average value) of the first sedimentation basin used during the experiment.

[0048]

[Table 4] (Note) * All units are mg / liter except pH. * All sugars are converted to glucose.

As the obligate anaerobic heterotrophic hydrogen-producing bacterium,
Clostridium butyricum ATCC859 was purchased from ATCC in the same manner as in Example 1, and the culture was expanded at 30 ° C. using a liquid medium of medium ATCC38. On the other hand, wild yeasts that are mixed and co-cultivated with Clostridium butyricum ATCC859
Purchased Mesh Nikowaia (Candida) Pulcherrima ATCC (Fungi / Yeasts) 7696 from TCC and added liquid medium of medium ATCC200 (Difco 0711) to p
Using the one adjusted to H6.0, the culture was expanded by a conventional method. Experimental conditions for hydrogen fermentation of sludge in the first settling tank * Days of culture: 10 days, * Culturing temperature: 30 ° C, * Organic load of hydrogen fermentation tank: 3.2 kg / m ³ /
Day, * pH value of the mixed culture solution in a dynamic equilibrium state: 6.
5, * Oxidation-reduction potential of the mixed culture was around -200 mV. Therefore, artificial pH adjustment and redox-potential control were not performed during the hydrogen fermentation experiment.

In the comparative experiment, first, 800 ml of the test sludge having the water quality shown in Table 4 was placed in a hydrogen fermentation tank having an effective volume of 1 liter.
Stick in. On the other hand, the heterotrophic hydrogen-producing bacterium Clostridium and the wild yeast mesh Nicowire (Mets
The genus chnikowia) was inoculated in an amount of 100 ml each as a bacterial cell liquid obtained by increasing culture with a predetermined culture solution, and the total amount was 1 liter. On the other hand, in a comparative control experiment, only 200 ml of the heterotrophic hydrogen-producing bacterium was inoculated into the culture broth to make the total amount 1 liter. For about a week from the start of the experiment, the culture was carried out at a temperature of 30 ° C. without supplying a substrate, and the culture was acclimated until the gas generation showed an increasing tendency.

Thereafter, the substrate was charged in a semi-continuous manner under a load condition of about ½ of the final load condition, and the load condition was gradually increased, and the operation was started under the final load condition after about one month. About 40 days after the start of the hydrogen fermentation experiment, data that can be regarded as a steady state was obtained, so about 50 days later, about 20 days later.
Daily, accurately weighed amount of hydrogen gas generated on alternate days, whereas to measure the total sugar content (in terms of glucose) seeking removed all sugar content in the fermentation digestive _{juices, mol · H 2 / mol ·} C 6 H 12 O ₆ was calculated. The results obtained from this comparative experiment are shown in FIG. In FIG. 2,-●-is (a) Clostridium +
Mesh Nicowire (pH 6.9 to 6.5) and-○-are (b) Clostridium alone (pH 6.4).

Experimental Results (1) As a result of the hydrogen fermentation experiment of the first settling tank sludge, hydrogen fermentation was clarified even if only the obligately anaerobic heterotrophic hydrogen-producing bacterium Clostridium butyricum ATCC859 was inoculated as a control experiment. ATP was deficient and the ergon reaction occurred, and considerable lower fatty acids were accumulated in the fermentation digestion liquid, and hydrogen fermentation was not completed. As a result, the hydrogen production was extremely small, and H ₂ /
C ₆ H ₁₂ O ₆ is only 1.5 to 2.5 mol,
The finding was that hydrogen production was practically impossible.

(2) In contrast, the biological hydrogen production method of the present invention, Clostridium + wild yeast (Metschni
hydrogen fermentation by mixed co-cultivation of kowia) is approximately 8-9m
high hydrogen production of _{ol · H 2 / mol · C} 6 H 12 O 6 was obtained. In addition, the removal rate of the total sugar amount in this experiment showed a high value of 90% or more during the experiment period. (3) From the results of the above-mentioned demonstration experiments, Clostridium is solubilized even in organic waste that contains high-concentration organic matter, such as raw sludge in the sedimentation basin, and also contains sulfides that are microbial poisons. Alternatively, wild yeasts (Metschnikowia sp., Etc.) produce excess ATP using low-molecular-weight sugars and / or lower fatty acids as substrates, and supply this ATP to the hydrogen production reaction of Clostridium sp. To complete hydrogen fermentation. It was proved that the above conjugate relation was clearly established.

[0054]

As described above, the specification and the first and second embodiments
As described and verified in detail in the above, the obligately anaerobic heterotrophic hydrogen-producing bacterium Clostridium and brewing yeast (mainly Saccharomyces) and / or wild yeast (mainly Candida) according to the present invention are subjected to obligate anaerobic conditions. It became possible to stably produce a large amount of hydrogen by carrying out mixed co-cultivation with. The establishment of this outstanding biological hydrogen production method has the following remarkable effects. (1) To produce hydrogen from various conventional organic wastes using Clostridium alone and under anaerobic conditions, ATP
However, the hydrogen production reaction was an absorption ergon reaction, and as a result, the hydrogen production amount was small, there was a problem in stability, and practical application was difficult, but the present invention enabled practical application.

(2) It was concluded that the heterotrophic hydrogen-producing bacterium of the present invention and the yeast for brewing can be co-cultivated, and as a result,
It has become possible to recycle surplus brewer's yeast, which has been difficult to effectively use or dispose of in the past, and, as verified in Example 1, wort with high contamination strength in wastewater from the beer manufacturing process. Mixed wastewater of squeezing wastewater and waste yeast wastewater,
It became possible to convert it into clean energy called hydrogen. This makes it possible to treat alcohol using brewing yeast, wastewater treatment from about 100 or more factories nationwide, and energy production. (3) The yeast for brewing and / or the wild yeast is originally AT
Not only is P productivity high, but it also has the property of coupling excess ATP with the ergonometric reaction of other microorganisms. By effectively utilizing this property, conventional anaerobic fermentation progresses significantly in the positive direction. However, as a result, the treated water quality of the fermented digestive juice is significantly improved.

[Brief description of drawings]

FIG. 1 is a graph showing the amount of hydrogen gas generated according to the number of days of semi-continuous hydrogen fermentation in Example 1.

FIG. 2 is a graph showing the amount of hydrogen gas generated according to the number of days of hydrogen fermentation in Example 2.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location C12R 1: 145 1:85) (C12P 39/00 C12R 1: 145 1: 645) (C12P 3 / 00 C12R 1: 145 1:85) (C12P 3/00 C12R 1: 145 1: 645)

Claims (3)

[Claims] 1. A method for biologically producing hydrogen from an organic substrate, which comprises an obligate anaerobic, heterotrophic, and hydrogen-producing bacterial group in the organic substrate, and a yeast. A biological hydrogen production method, which comprises mixing and cultivating A. under an anaerobic condition. 2. The mixed culture has a pH of 5.5-6.
5. The biological hydrogen production method according to claim 1, wherein the redox potential is −150 to −250 mV. 3. The biological hydrogen production method according to claim 1, wherein the organic substrate is organic wastewater and organic waste including sludge.