WO2017183110A1

WO2017183110A1 - Biogas production method and device

Info

Publication number: WO2017183110A1
Application number: PCT/JP2016/062383
Authority: WO
Inventors: 栄城米澤
Original assignee: 株式会社ヨネクニ
Priority date: 2016-04-19
Filing date: 2016-04-19
Publication date: 2017-10-26
Also published as: JPWO2017183110A1; JP6579534B2

Abstract

[Problem] To generate a biogas from plant biomass such as wood, bamboo, etc., without physical and chemical treatments, which are essential in conventional acid-hydrolysis techniques and enzymatic saccharification techniques, and collect the biogas in a gas state from a solid state. [Solution] In the present invention, an imperfect fungus of the genus Wickerhamomyces is fermented with a carbon source to generate a biogas. In this biogas generation step, the imperfect fungus of the genus Wickerhamomyces is used. The life cycle of the imperfect fungus of the genus Wickerhamomyces is artificially changed between a filamentous fungus/basidiomycete-like life cycle and a yeast-like life cycle. The biogas is generated by repeating steps of: seeding the imperfect fungus of the genus Wickerhamomyces onto the carbon source and controlling culture conditions, thereby producing glucose from cellulose; and generating a biogas such as ethanol, etc., from the glucose. Since the biogas can be collected in a gaseous state directly from the solid carbon source, the cost to collect the component is reduced.

Description

Biogas production method and apparatus

The present invention relates to a method for producing biogas from plant biomass using fungi. In particular, the present invention relates to an apparatus and method for producing biogas from plant biomass using imperfect bacteria belonging to the genus Wickerhamomyces.

Effective utilization of unused plant biomass resources is required. In the prior art, biomass ethanol production using starch raw materials such as corn and potato, which are considered to be relatively easy to use among plant biomass resources, and sugar raw materials such as sugar cane is being promoted.
However, there are examples of plant biomass resources that have not been used yet, for example, lignocellulosic biomass resources such as thinned wood, construction waste, and rice straw, are used as a heat source by burning. It is relatively difficult to produce bioethanol from cellulosic plant biomass resources to obtain biofuel.

The fungus existing in nature that directly decomposes lignocellulosic biomass resources to produce ethanol is not yet generally known. Therefore, in the prior art, a cycle is known in which woody cellulose is subjected to physical treatment or chemical treatment to be once decomposed into glucose, and then alcohol is obtained from yeast based on glucose. As a widely known conventional technique, an acid hydrolysis method and an enzymatic saccharification method are known.

First, in the acid hydrolysis method, ethanol is obtained in a cycle as shown in the upper diagram of FIG.
As a 1st process, the delignification process is performed with respect to woody with a physical and chemical method with a solid phase, and the cellulose which is easy to process is obtained. As a 2nd process, a strong acid is added and hydrolyzed with respect to a cellulose, and glucose is obtained. Since the hydrolysis is performed by adding a strong acid, the treatment state becomes a liquid phase at this stage. As a third step, yeast is seeded using liquid phase glucose as a base material and subjected to alcohol fermentation to obtain an alcohol-containing fermentation broth. This state also remains a liquid phase.

Next, in the enzymatic saccharification method, ethanol is obtained in a cycle as shown in the lower diagram of FIG.
As a 1st process, the delignification process is performed with respect to woody with a physical and chemical method with a solid phase, and the cellulose which is easy to process is obtained. In the second step, cellulose (β-1,4-glucan) is further decomposed by adding physical / chemical treatment to cellulose. For example, there can be various methods such as a method of applying ozone water, a method of applying supercritical water, and a method of explosion. It becomes a liquid phase at this stage. In the third step, glucose is obtained using cellulase, which is a kind of enzyme, using treated cellulose (β-1,4-glucan) as a base material. Cellulase is an enzyme that hydrolyzes the glycosidic bond of cellulose (β-1,4-glucan) and can obtain glucose. This stage also remains in the liquid phase. As a 4th process, yeast is seed | inoculated and made into alcohol fermentation using liquid phase glucose as a base material, and an alcohol containing fermented liquid is obtained. This state also remains a liquid phase.

Next, in the prior art, a literature example disclosing a cycle from decomposition of woody cellulose to ethanol fermentation using a fungus without using a physical process / chemical process is small, for example, WO2012 / No. 2164990 exists. This discloses a method for producing ethanol by degrading woody cellulose using basidiomycetes belonging to the genus Phlebia. The inventor Ichiro Kamei et al. Of the application described that basidiomyceae belonging to the genus Phlebia not only have lignin resolution and polysaccharide saccharification ability, but also have the ability to produce ethanol from sugar, and not only glucose but also xylose. Disclosed that it has the ability to produce ethanol even when used as a carbon source, and further, after performing a pretreatment step of fermenting basidiomycetes belonging to the genus Phlebia together with a lignin-containing carbon source under aerobic conditions, Under semi-aerobic conditions or anaerobic conditions, ethanol production efficiency can be further increased by further fermenting basidiomycetes belonging to the genus Phlebia together with a carbon source and performing ethanol fermentation using the carbon source as a substrate. Disclosure.

As shown above, the conventional acid hydrolysis and enzymatic saccharification methods require multiple physical and chemical processes, as well as biodegradation processes of enzymes and yeast fermentation processes. It was known as a method for obtaining bioethanol from wood. In addition, there are few reports on how to obtain bioethanol from woody materials such as thinned wood using fungi.

WO2012 / 2164990

Here, the acid hydrolysis method for producing ethanol using the lignocellulosic biomass resource as a raw material, the enzymatic saccharification method, and the method disclosed in WO2012 / 2164990 in the above-mentioned conventional technology have the following problems. there were.

First, the conventional acid hydrolysis method has the following problems.
In the conventional acid hydrolysis method, it is necessary to subject the wood to delignification by a physical / chemical method as the first step. However, this delignification treatment is not easy and costly. It requires energy for chemical and chemical treatment.
Further, in the conventional acid hydrolysis method, as a second step, it is necessary to add a strong acid to the cellulose to hydrolyze. However, the hydrolysis by the addition of the strong acid is costly, and physical / chemical The process requires energy, and physical and chemical costs are required to treat the strong acid after the reaction. In addition, there is a problem that it is difficult to control the decomposition rate of cellulose. In addition, glucose may be decomposed with a strong acid.
In addition, in the conventional acid hydrolysis method, as a third step, it is necessary to inoculate yeast using liquid phase glucose as a base material for alcohol fermentation, but the delignification treatment or the second step in the first step is required. In the process of hydrolysis with strong acid in the process, substances that adversely affect the yeast in the liquid phase are mixed, so alcohol fermentation is inhibited as it is, so it is necessary to wash glucose, improving cost, energy Consumption increased.

Next, the conventional enzymatic saccharification method has the following problems.
In the conventional enzymatic saccharification method, as a first step, it is necessary to perform delignification treatment on wood by a physical / chemical method. However, as pointed out above, this delignification treatment is not easy and costly. And requires energy for physical and chemical treatment.
In the conventional enzymatic saccharification method, as a second step, it is necessary to add a physical / chemical treatment to cellulose to obtain a cellulose that has been further decomposed. Both the method of acting and the method of blasting require physical and chemical costs, and the reaction kettle and processing equipment are special and the manufacturing equipment is also expensive.
In addition, in the conventional enzymatic saccharification method, as a fourth step, it is necessary to inoculate yeast using liquid phase glucose as a base material for alcohol fermentation, but as described above, the delignification treatment in the first step Alcohol fermentation that occurs in the hydrolysis treatment with a strong acid in the second step or the like has resulted in an increase in cost and an increase in energy consumption due to the removal of inhibitors.

Next, the method described in WO2012 / 2164990, which obtains bioethanol directly from a woody material using basidiomycetes belonging to the genus Phlebia, has the following problems.
The method of directly obtaining bioethanol from wood using basidiomycetes belonging to the genus Phlebia does not involve physical and chemical treatments that are essential procedures for conventional acid hydrolysis and enzymatic saccharification methods. Is a remarkable technology.
However, the method of obtaining bioethanol directly from wood using basidiomycetes belonging to the genus Phlebia is that the phase in which basidiomycetes produce bioethanol is a liquid phase. Ethanol is highly hydrophilic, and once it is in a liquid phase, it cannot be isolated without a physical distillation step. The distillation process caused an increase in cost and energy.
The problem of isolating ethanol from liquid-phase bioethanol is a problem common to the acid hydrolysis method and enzymatic saccharification method described above.

Moreover, it is the point of the basidiomycete which belongs to Phlebia genus, and the combination of the wood which can be utilized. In this publication, saccharides that can be used as a carbon source include hexoses such as glucose, mannose, galactose and fructose, monosaccharides such as pentoses such as xylose and arabinose, and disaccharides such as cellobiose. The plant biomass material is not particularly limited. However, basidiomycetes are so-called mushrooms, and it is widely known that mushrooms grow by type and the types of parasitic wood are limited. Mushrooms are cultivated in raw wood cultivation using compatible wood logs or in fungus bed cultivation using a fungus bed and rice bran mixed with nutrient sources such as bran and rice bran Therefore, it is considered that compatibility with so-called trees and fungus beds which are pulverized products is good.
Although mushrooms are parasitic on well-matched trees, in general, only a few mushrooms are said to grow in bamboo, cedar and cypress forests. In recent years, invasion of bamboo into economic forests has become a problem, but bamboo is said to be incompatible with basidiomycetes.
As described above, the bioethanol production method using plant biomass using basidiomycetes belonging to the genus Phlebia as a raw material may limit the types of plant biomass that can be used.

Next, Non-Patent Document 1 discloses bioethanol production using Wickerhamomyces anomalus. From the viewpoint of the disclosed production process, as a first process, a carbon source is hydrolyzed to convert cellulose and the like into glucose. It is premised on the process of decomposing, and enormous amounts of energy and chemicals are required, and the process is in the liquid phase. Non-Patent Document 1 is a method in which Wickerhamomyces anomalus is seeded on glucose and fermented with ethanol. The process of producing ethanol from glucose is known as a fermentation means using other bacteria such as yeast, and the use of Wickerhamomyces anomalus in Non-Patent Document 1 is only an alternative means of producing ethanol from glucose. There is nothing.
In addition, since the nonpatent literature 1 presupposes the hydrolysis process of the carbon source in a liquid phase, the utilization stage of Wickerhamomyces anomalus is also a liquid phase. Ethanol is highly hydrophilic, and once it is in a liquid phase, it cannot be isolated without a physical distillation step. The distillation process increases costs and energy.

Next, in Non-Patent Document 2, the overall purpose is ethanol fermentation from wheat straw, but the part where Wickerhamomyces is involved is how to preserve the carbon source of wheat straw that can be harvested in large quantities before the winter season. It is a preservation process, and it is not involved at all in the fermentation process for producing ethanol from wheat straw as a carbon source. In Non-patent Document 2, winter preservation using Wickerhamomyces anomalus called ISP treatment is performed, but in the spring, Wickerhamomyces anomalus is separated and removed before ethanol fermentation. Ethanol fermentation itself consists of acid treatment, It is fermentation by decomposition treatment and DUET enzyme seeding in liquid phase, Wickerhamomyces anomalus is not involved in fermentation, and fermentation itself is fermentation in liquid phase.

Therefore, the present invention has been made in view of the above problems, and is a plant biomass that is used as a raw material in a form that does not involve physical and chemical treatment, which is an essential procedure in conventional acid hydrolysis methods and enzymatic saccharification methods. The objective is to obtain biogas such as ethanol and ethyl acetate directly from
In addition, the present invention aims at generating biogas from plant biomass and recovering it from the solid phase in the gas phase, and in the liquid phase as in the conventional acid hydrolysis method, enzymatic saccharification method and non-patent literature. The purpose is to reduce the recovery cost by recovering the biogas in the gas phase directly from the solid-state carbon source.

The inventor of the genus Wickerhamomyces has not only cellulose-degrading ability, lignin-degrading ability, and polysaccharide saccharification ability, but also ability to produce ethanol from sugar, and further produces ethyl acetate via ethanol. I found that I have the ability to do. In general, this incomplete bacterium of the genus Wickerhamomyces is known to rot processed foods, and various studies and reports have been made on control methods for suppressing growth. However, the present inventor has developed and constructed for the first time a technology that makes effective use of this bacterium, generates a useful biogas in various phases using solid biomass, and easily recovers it.

For example, as an incomplete bacterium belonging to the genus Wickerhamomyces, there is Pichia.
An imperfect bacterium belonging to the genus Wickerhamomyces transitions between a life cycle different from a filamentous fungi-like life cycle and a yeast-like life cycle. The present invention focuses on the properties of imperfect bacteria belonging to the genus Wickerhamomyces.

The biogas production method of the present invention is a biogas production method including a biogas production step of producing biogas by culturing an incomplete bacterium belonging to the genus Wickerhamomyces together with a carbon source, and belongs to the genus Wickerhamomyces An incomplete bacterium transitions between a life cycle of a filamentous fungus basidiomycetous life cycle and a life cycle different from a yeast-like life cycle, and the biogas generation step converts the incomplete bacterium belonging to the genus Wickerhamomyces to a filamentous fungus basidiomycetes A first step of degrading cellulose of the carbon source in a solid phase by culturing in a life-like life cycle, and a first step of culturing incomplete bacteria belonging to the genus Wickerhamomyces in a yeast-like life cycle In the solid state, the glucose in the solid state generated from the carbon source in the solid state is decomposed to produce methanol, ethanol, any of their esters, or a mixture thereof. A second step of generating a biogas, wherein the filamentous basidiomycetous life cycle and the yeast-like life cycle of the incomplete bacterium belonging to the genus Wickerhamomyces are transitioned in the first step and the second step. And a process for producing the biogas in a gas phase state directly from the carbon source without going through a liquid phase.
Biogas is generated by using the carbon source as a material through the first step and the second step, and further, the first step and the second step are continuously repeated alternately. In the process, the biogas in the gas phase can be produced directly from the carbon source in the solid phase without going through the liquid phase.

Here, the condition for transition from the first step to the second step is a yeast-like life cycle activity condition for activating the yeast-like life cycle of an incomplete bacterium belonging to the genus Wickerhamomyces, The condition for transition from the step 2 to the first step is a filamentous fungus basidiomycetous life cycle activation condition for activating the filamentous fungus basidiomycetous life cycle of the incomplete bacterium belonging to the genus Wickerhamomyces, By appropriately switching between the yeast-like life cycle activity condition and the filamentous fungus basidiomycet-like life cycle activity condition as culture conditions, the first step and the second step are alternately switched while the biogas Proceed with generation.
For example, the yeast-like life cycle activity conditions are normal temperature and anaerobic conditions, and the filamentous fungi-like life cycle activity conditions are normal temperature and aerobic conditions.

The above biogas production method realizes solid phase fermentation. Solid-phase fermentation is a process in which the carbon source is a powder or crushed solid phase, and the biogas generation step generates biogas in a gas phase from a solid-state carbon source. It is a fermentation that generates biogas directly from a carbon source. Various technical effects can be obtained by realizing the solid-phase fermentation technique using incomplete bacteria belonging to the genus Wickerhamomyces.

The first technical effect of the solid-phase fermentation technology is the elimination of adverse effects on the fermentation cycle caused by alcohol components produced during the production process, which can be an inhibiting factor of the fermentation cycle. The alcohol component produced in the production process has a high bactericidal power. In the case of liquid phase fermentation, the alcohol component dissolves in the liquid due to its high hydrophilicity and is present in the system. Therefore, if the alcohol concentration exceeds the growth limit of the bacteria, the growth of the bacteria may be suppressed. . However, since the solid fermentation technology has been completed in the present invention, alcohols harmful to the bacteria do not move to the liquid phase, but volatilize directly from the solid phase carbon source and are discharged out of the system as a gas phase. It is not a factor that inhibits the fermentation of fungi.

The second technical effect of the solid phase fermentation technique is a reduction in the recovery cost of the alcohol component and ester component. In the case of liquid phase fermentation, the upper limit of the concentration of alcohol that can be produced is about 15%, and a concentration step such as distillation is essential for industrial use. However, if the solid state fermentation technique of the present invention is used, the fermentation product is produced in a gaseous state, so that it can be directly concentrated and liquefied easily by an aggregating device such as a cooling trap. In addition, by controlling the cooling temperature, even if the biogas is produced in the mixed state of alcohol component and ester component, the advantage is that it can be easily selectively concentrated for each target component by setting the temperature of the aggregator such as a cooling trap. There is also.

The incomplete bacterium belonging to the genus Wickerhamomyces is a bacterium that exists in nature, but no solid-phase fermentation of alcohols and esters from a carbon source as in the present invention has been reported. In the natural state, incomplete bacteria belonging to the genus Wickerhamomyces basically have a slow growth rate, but they cannot catch up with the growth of lactic acid bacteria, which are universal resident bacteria. When inoculated, the growth rate is inevitably inferior, and the incomplete bacteria belonging to the genus Wickerhamomyces are inhibited by fermentation products such as lactic acid, so the potential of the incomplete bacteria belonging to the genus Wickerhamomyces is fully active. It is thought that it was not made. In other words, imperfect bacteria belonging to the genus Wickerhamomyces can fully exert glucose production reactions and alcohol / ester production reactions while transitioning between the filamentous fungi-like life cycle and the yeast-like life cycle. However, it is thought that the conditions and environment did not match naturally. Also, in the natural environment, even if imperfect bacteria belonging to the genus Wickerhamomyces overcome lactic acid bacteria and proliferate predominately under a certain carbon source, the fungal basidiomycetous life cycle under aerobic conditions in the atmosphere After glucosylation of cellulose, there is no natural switching to anaerobic conditions, and imperfect bacteria belonging to the genus Wickerhamomyces do not transition to the yeast-like life cycle. This is probably because the portion changed to glucose was not further decomposed, or the change proceeded only very slowly.

In the present invention, by artificially repeating the life cycle of imperfect bacteria belonging to the genus Wickerhamomyces, its potential is exerted, and the change from solid phase carbon source cellulose to glucose and glucose to ethanol is repeated. It is possible to repetitively produce a gas phase biogas directly from a solid state carbon source.

Here, the first first step may be started from an aerobic condition similarly to the first step repeatedly appearing thereafter, but there may be a device that starts from an anaerobic condition instead of starting from an aerobic condition. It is a device that artificially prevents lactic acid bacteria having a wide antibacterial spectrum mixed in a carbon source from proliferating predominately. That is, in the alternating repetition of the first step and the second step described above, in the first first step, the culture condition is not changed to the filamentous fungus-like basidiomycetous life cycle activity condition. From the above, the yeast-like life cycle activity conditions are normal temperature and anaerobic conditions, suppress the growth of lactic acid bacteria mixed in the carbon source, and incomplete bacteria belonging to the genus Wickerhamomyces produce glucose from cellulose as the carbon source. However, since it is under the yeast-like life cycle activity condition, the life cycle is controlled to naturally transit to the yeast-like life cycle and to transit to the second step.
By starting from anaerobic conditions at the time of the first first step, the growth of aerobic lactic acid bacteria can be suppressed, and at the time of the first first step, incomplete bacteria belonging to the genus Wickerhamomyces are propagated from lactic acid bacteria. Can become dominant. Since the predominance of imperfect bacteria belonging to the genus Wickerhamomyces that subsequently transitioned to the yeast-like life cycle increases in the second step thereafter, the filamentous fungus basidiomycetous life cycle at the time of transition to the first step thereafter Even under active conditions, that is, aerobic conditions, imperfect bacteria belonging to the genus Wickerhamomyces are sufficiently dominant, and fermentation can be continued without losing lactic acid bacteria.

In the repetition of the first step and the second step, switching from the first step to the second step is performed at an arbitrary timing after the rate of change of the cellulosic surface of the carbon source to glucose has passed the peak. Yes, the switching from the second step to the first step may be performed at an arbitrary timing after the rate of change from glucose to biogas which is ethanol, ethyl acetate, or a mixture thereof passes the peak.

The carbon source in the biogas production apparatus of the present invention can widely use plant biomass materials including wood, rice straw, bamboo and the like. Lignocellulose-based plant biomass materials can be widely used. As will be described later, the inventor of the present invention uses the biogas production method of the present invention, so that biogas in a solid phase using bamboo flour, which is generally considered difficult to be fermented among plant biomass materials, as a carbon source. Generated successfully.

The biogas produced in the biogas production method of the present invention may include any of alcohols and esters thereof, volatile organic acids and esters thereof, or any combination thereof. Examples of alcohols include methyl alcohol and ethyl alcohol. As a possibility, caproic acid, caprylic acid, capric acid and the like can be generated as other alcohols such as butyl alcohol and amyl alcohol and volatile organic acids. Esters may include the above-described alcohol esters and volatile organic acid esters.
For example, ethanol has already attracted attention as a bioethanol fuel, and ethyl acetate gas is also in great demand as various industrial raw materials.

The present inventor, as a material for realizing the biogas production method of the present invention, generates biogas from a carbon source, which includes imperfect bacteria belonging to the genus Wickerhamomyces and a carrier carrying the imperfect bacteria belonging to the genus Wickerhamomyces We have also succeeded in producing an inoculum for this purpose.

The biogas generating apparatus of the present invention is preferably an apparatus that can be controlled so as to maintain the culture conditions of the incomplete bacteria belonging to the genus Wickerhamomyces described above. Specifically, a culture tank for culturing the carbon source and the incomplete bacteria belonging to the genus Wickerhamomyces sowed on the carbon source, and the culture conditions of the culture tank are the yeast-like life cycle activity condition and the filamentous fungus basidiomycete It is assumed that culture condition setting means that can be switched under the fungus-like life cycle activity conditions and biogas recovery means that recovers the generated biogas are provided. A configuration with a carbon source stirring device is also preferred.

According to the biogas production method of the present invention, an incomplete bacterium belonging to the genus Wickerhamomyces is effectively used, and various biomass is used to decompose and ferment useful biogas in a solid phase, Can be generated and easily recovered.
Since it decomposes and ferments in the solid phase, the carbon source is used as a solid phase carbon source such as powder or crushed material, and the biogas generation process generates biogas in the gas phase directly from the solid state carbon source. Therefore, it is possible to generate a biogas in a gas phase directly from a carbon source in a solid phase without going through a liquid phase. Solid-phase fermentation eliminates adverse effects on the fermentation cycle caused by alcohol components generated in the biogas production process, and even when biogas is produced in a mixed state of alcohol and ester components, agglomerates such as cooling traps Selective concentration can be easily performed for each target component by setting the temperature of the apparatus.

It is a figure explaining the life cycle regarding the imperfect bacteria which belong to the genus Wickerhamomyces used with the biogas manufacturing apparatus and manufacturing method of this invention. BRIEF DESCRIPTION OF THE DRAWINGS It is the figure which showed simply the biogas manufacturing apparatus of this invention concerning Example 1, and the biogas manufacturing method of this invention performed in the said apparatus. It is the figure which showed simply the structure of the laboratory instrument for verifying the biogas manufacturing method of this invention. It is a figure which shows the enlarged photograph of the sample of the bamboo powder used for verification experiment. It is a figure which shows the collection | recovery result of the biogas in verification experiment. It is a figure which shows the time-sequential change of the kind of biogas to generate | occur | produce. It is the figure which showed the mode near the bottom face of the flask for solid-phase fermentation which assumed the biogas production | generation part 110 immediately after completion | finish of experiment, and the figure which shows the culture result using a culture medium. It is the figure which showed simply the biogas manufacturing apparatus of this invention concerning Example 2, and the biogas manufacturing method of this invention performed in the said apparatus. It is the figure which showed the culture | cultivation result using the figure which showed simply returning from the 1st 1st process to the 1st process through the 2nd process first, and a culture medium. It is the figure which showed simply that it transfers to a 2nd process again from the state which returned to the 1st process again, and a figure which shows a mode that it repeats with a 1st process and a 2nd process after that. It is the figure which showed the change of the carbon source simply. In the prior art, it is a figure explaining the acid hydrolysis method and enzyme saccharification method which obtain alcohol using wood type cellulose as a raw material.

Hereinafter, the biogas production method of the present invention and examples of inoculum for producing biogas will be described with reference to the drawings.
In addition, the following Examples are examples and the content of this invention is not limited to the specific content of an Example.

The present invention controls the activity of imperfect bacteria belonging to the genus Wickerhamomyces, controls the transition of the different life cycles of the filamentous fungi-like life cycle and the yeast-like life cycle, and performs solid-state fermentation using a carbon source as a raw material. It is possible to produce biogas containing alcohols and esters.
The incomplete bacterium belonging to the genus Wickerhamomyces used in the present invention can undergo the following solid-state fermentation cycle by transitioning between a life cycle of a filamentous fungus-like basidiomycetous life cycle and a yeast-like life cycle.

FIG. 1 illustrates the life cycle of imperfect bacteria belonging to the genus Wickerhamomyces used in the biogas production method of the present invention.
As shown in FIG. 1, the filamentous basidiomycete-like life cycle is easily expressed in a cellulose-rich state under aerobic conditions in the progress of decomposition of the carbon source, and the progress from cellulose to glucose to alcohols is promoted. The life cycle tends to be expressed in a glucose-rich state under anaerobic conditions, and progresses from glucose to alcohols to esters.

The inventor has invented a method of transitioning between two life cycles of incomplete bacteria belonging to the genus Wickerhamomyces to advance the decomposition rate of the carbon source. The generation of biogas should start from the filamentous basidiomycetous life cycle, as long as the decomposition of cellulose into glucose by the fungal basidiomycetous life cycle and the production of glucose to biogas by the yeast-like life cycle is performed. Biogas production can be seen when transitioning to a yeast-like life cycle. Furthermore, as shown in FIG. 1, the transition control from the filamentous basidiomycetous life cycle to the yeast-like life cycle and the transition control from the yeast-like life cycle to the filamentous basidiomycetous life cycle are artificially manipulated. Thus, it is possible to repeatedly generate biogas by repeatedly performing the decomposition progress of the carbon source.
Thus, the technology of generating biogas from a carbon source in which the transition between two life cycles of imperfect bacteria belonging to the genus Wickerhamomyces is artificially controlled is an unprecedented breakthrough.

FIG. 2 simply shows the biogas production apparatus according to the first embodiment and the biogas production method of the present invention performed in the apparatus.
FIG. 2A simply shows the configuration of the biogas production apparatus 100 used in the present invention according to the first embodiment.
As shown in FIG. 2 (a), a solid phase carbon source can be appropriately charged by a carbon source charging means 120 with a culture tank 110 as a center. Moreover, the culture condition setting means 130 is provided and the culture conditions in the culture tank 110 can be controlled. In this embodiment, the culture condition is controlled by switching between the anaerobic condition and the aerobic condition, and the culture condition setting means 130 controls the valve 141 of the nitrogen supply means 140. The biogas recovery means 160 is a device that receives the biogas such as methanol, ethanol, ethyl acetate, etc. in the gas phase generated from the cellulose through glucose through the culture tank 110, and recovers it in the gas phase. is there.

FIG. 2B shows how the first step and the second step are performed as the biogas production method of the present invention.
The first step is a step of culturing incomplete bacteria belonging to the genus Wickerhamomyces together with a solid-state carbon source to produce glucose from the carbon source cellulose.
In the second step, subsequent to the first step, incomplete bacteria belonging to the genus Wickerhamomyces are cultured to decompose the solid-phase glucose produced in the first step to decompose gas phase methanol and ethanol. , Producing biogas which is any of ethyl acetate or a mixture thereof.
By performing the first step and the second step, a biogas in a gas phase state is directly produced from a carbon source in a solid phase state without passing through a liquid phase.

When the culture condition setting means 130 makes a transition from the first step to the second step, the yeast-like life cycle activity condition, that is, the inside of the culture tank 110 is maintained at room temperature and anaerobic conditions, so that nitrogen is supplied as necessary. Control of opening the valve 141 of the means 140 is performed to maintain an appropriate anaerobic condition.

In this example, the first first step is an example starting from anaerobic conditions. This is because lactic acid bacteria mixed in the carbon source may proliferate predominantly under aerobic conditions. Even under anaerobic conditions, an incomplete bacterium belonging to the genus Wickerhamomyces is producing yeast from cellulose as a carbon source, but it is a yeast-like life cycle activity condition, so that the filamentous basidiomycetous life cycle will eventually become yeast. It will naturally transition to the life cycle, and will transition to the second step.

Hereinafter, the biogas production method of the present invention will be demonstrated by experiments.
[Apparatus configuration used in the demonstration experiment]
FIG. 3 is a diagram showing a configuration of a laboratory instrument for verifying the biogas production method.
The flask for solid phase fermentation assumes a culture tank 110.
The flask for solid-phase fermentation encloses bamboo powder as a carbon source, nitrogen-distilled at 70 ° C. for 1 week, and seeded with imperfect bacteria belonging to the genus Wickerhamomyces.
The culture condition setting means 130 was omitted by manually operating the experimental instrument.
The oxygen cylinder imitates the oxygen supply means 150, and the nitrogen cylinder imitates the nitrogen supply means 140. The oxygen cylinder used in the latter stage of the demonstration experiment is used when the inside of the flask for solid-phase fermentation assumed to be a culture tank is in an aerobic condition, but even an air cylinder into which sterilized air is introduced. good.
Anaerobic conditions are maintained by supplying nitrogen gas from a nitrogen cylinder at 2 ml / min.
Aerobic conditions are maintained by supplying oxygen gas or air from an oxygen or air cylinder at 2 ml / min.
The biogas recovery device 160 was replaced with a cool trap device. The cool trap device is pre-cooled at −20 ° C. in which a glass ball is put in a gas trap bottle and piped.
In addition, it is an experiment which assumed the basic structure of the biogas manufacturing apparatus of this invention by the structure of said experimental instrument.

[Bacteria used in the demonstration experiment]
Hereinafter, incomplete bacteria belonging to the genus Wickerhamomyces used in the demonstration experiment were identified and confirmed.
For the analysis of the bacteria, the microorganism used in the demonstration experiment was commissioned to Techno Suruga Lab Co., Ltd., which undertakes the microorganism identification test, and the microorganism identification test was requested. As a specific identification method, microbial community structure analysis was performed using a denaturing gradient gel electrophoresis (DGGE) method. The DGGE method is an electrophoresis method that separates double-stranded DNA fragments having the same length based on differences in base sequences. DNA was extracted from the excised band, and the DNA type was analyzed while confirming the purity of the band by performing DGGE again using the product amplified by PCR using this as a template. The 7-band 28S rDNA partial base sequence and the 1-band 16S rDNA partial base sequence obtained by DGGE analysis were determined, and a simple lineage analysis was performed based on these results to determine the belonging taxon of the bacterial group derived from each band.

Cycle sequence: Big Dye Terminator v3.1 Cycle Sequencing Kit
(Applied Biosystems, CA, USA)
Primer used: DGGE band sequencing kit for analysis of the bacterial v3 region
(DS-0001) (TechnoSuruga Laboratory Co., Ltd, Shizuoka)
Lac1, Lac3, Lac2 (for Lactobacillales eye amplification) Lac1, Lac 3 are mixed in equal amounts Sequence: ABI 3130xl Genetic Analyzer System (Applied Biosystems, CA, USA)
Sequencing: ChromasPro 1.4 (Technelysium Pty Ltd., Tewantin, AUS)
Homology search and simple molecular phylogenetic analysis: Software Apollon 3.0 (Techno Suruga Lab, Shizuoka)
Database: International nucleotide sequence database (GenBank / DDBJ / EMBL)
Apollon DB-BA10.0 (Techno Suruga Lab Shizuoka)
Apollon DB-FU (D1 / D2) 8.0 (Techno Suruga Lab Shizuoka)
Hereinafter, the identification result regarding the microbe used for the demonstration experiment as an incomplete microbe belonging to the genus Wickerhamomyces used in the biogas production method of the present invention is shown.
Two test samples, test category 1 and test category 2, were prepared and double-checked.

[Analysis of test category 1 sample]
Test 1 Apollon DB-FU (D1 / D2) 8.0 search results for the homology between the base sequence and the top 15 strains

Test 2 Homology between base sequence and top 20 strains GenBank / DDBJ / EMBL international base sequence database search results

[Analysis of test category 2 samples]
Test 1 Homology between base sequence and top 15 strains Apollon DB-FU (D1 / D2) 8.0 search results

As a result of homology search for Apollon DB-FU (D1 / D2) in [Table 1] and [Table 3], the 28S rDNA base sequence derived from the band showed the highest homology to 28 rDNA derived from Wickerhamomyces arborarius. It was.
Further, in the results of the GenBank / DDBJ / EMBL international base sequence database search in [Table 2] and [Table 4], high homology was shown to 28S rDNA such as Wickerhamomyces anomalus.
Judging all of them, it was concluded that the base sequence derived from the band was included in the 28S rDNA cluster of the genus Wickerhamomyces.
As a result, it was concluded that the bacteria used in the demonstration experiment were incomplete bacteria belonging to the genus Wickerhamomyces.

The incomplete bacteria belonging to the genus Wickerhamomyces used in the present invention belong to the genus Wickerhamomyces, and when cultured with a carbon source under solid-phase culture conditions, the filamentous fungi-like life cycle and yeast-like life cycle are different. It is not particularly limited as long as it is a bacterium that changes the life cycle and generates a biogas containing alcohols and esters thereof, volatile organic acids and esters thereof by solid phase fermentation using a carbon source as a raw material.
The inventor used the identified strain of the same strain as that of the incomplete strain belonging to the genus Wickerhamomyces.

[Carbon source used in the demonstration experiment]
The incomplete bacteria belonging to the genus Wickerhamomyces used in the present invention can transition between a life cycle of a filamentous fungus-like basidiomycetous life cycle and a life cycle different from a yeast-like life cycle, and therefore the carbon source is not particularly limited.
For example, the plant biomass material includes lignocellulosic biomass and the like, and may be a tree biomass material or a grass biomass material. Examples of the woody biomass material include woods (including building waste materials, thinned woods, etc.) derived from trees such as conifers, broadleaf trees, gymnosperms, or their bark, sawdust, leaves, mushroom waste fungus beds, and the like. Examples of grass-based biomass materials include materials derived from rice, wheat, corn, sugarcane, bamboo, Japanese pampas grass, and the like, for example, residues generated during harvesting and processing of agricultural products.

Plant biomass materials including lignocellulosic biomass such as hardwood wood, rice straw, bamboo, etc. are composed of hemicellulose consisting of pentoses such as xylose and hexoses such as glucose, and glucose. Contains a lot of cellulose. That is, as the carbon source of the biogas production method of the present invention, cellulose and hemicellulose can be used, but of course, saccharides that have been further decomposed, that is, hexoses such as starch, glucose, mannose, galactose, and fructose, xylose Monosaccharides such as pentose such as arabinose and disaccharides such as cellobiose can also be used as the carbon source.
In the present invention, the carbon source is directly used as a material in the solid phase, and gas phase biogas is generated. Therefore, the physical treatment and chemical treatment required for the use of the conventional lignocellulose-based carbon source are used. Is unnecessary.
The following demonstration experiment was conducted using bamboo powder, which is considered to be relatively difficult to use biomass.
FIG. 4 is an enlarged photograph of a sample of bamboo powder used in the demonstration experiment.

[Solid-phase fermentation process and culture conditions in the demonstration experiment]
The biogas production apparatus and production method of the present invention were verified by the following solid phase fermentation process and culture conditions.
First, in the biogas production method according to Example 1 of the present invention, an experiment was performed to demonstrate biogas production by transition to the first step and the second step.
・ Solid-phase fermentation process: Bamboo powder remains in solid phase and seeded with imperfect bacteria belonging to the genus Wickerhamomyces ・ Culture conditions: maintained at room temperature and anaerobic conditions

In addition to the solid-state culture conditions of the solid phase, the solid state of the carbon source or powdered body and the anaerobic conditions, in addition to the activity of natural lactic acid bacteria already mixed in the carbon source As described below, if sufficient anaerobicity is maintained, lactic acid bacteria are aerobic bacteria, so if the experiment is conducted in consideration of the activation being inhibited, The above-mentioned culture conditions satisfy the culture conditions in the solid phase.

As a carbon source, bamboo was used as a granular or powder solid phase. In the biogas production method of the present invention, the material may be only a carbon source, but it is not excluded to contain it together with necessary components such as a nitrogen source and inorganic salts as necessary.
Inoculation with bacteria is performed by inoculating an appropriate medium in a solid phase with incomplete bacteria belonging to the genus Wickerhamomyces. Here, bacteria such as lactic acid bacteria, other filamentous fungi, basidiomycetes, and yeasts are prevented from being mixed.
The culture temperature of the fungus is preferably an ordinary temperature of 25 ° C. to 35 ° C., and the culture time can be about 24 hours to 2000 hours. In the demonstration experiment, the culture temperature was 30 ° C. and the culture time was 1440 hours (60 days).

The culture of the bacteria shall be anaerobic culture. Specifically, anaerobic culture refers to culturing a medium inoculated with imperfect bacteria belonging to the genus Wickerhamomyces without substantially aeration with the outside air. In the present invention, anaerobic conditions refer to culture conditions that are substantially free of free enzyme.For example, imperfect bacteria belonging to the genus Wickerhamomyces and a medium are contained in a container, and the atmosphere in the container is filled with nitrogen gas or the like. Examples include culture conditions in which the enzyme is not substantially contained by substitution or the like, and the culture is performed in a state where the inside of the container and the outside air are not substantially vented.

As will be described later in the discussion, the biogas production method of the present invention controls to inhibit the activation of natural lactic acid bacteria mixed in the carbon source by culturing under anaerobic conditions. Lactic acid bacteria are aerobic bacteria, and under aerobic conditions, the fertility is superior to incomplete bacteria belonging to the genus Wickerhamomyces, and the reproduction of incomplete bacteria belonging to the genus Wickerhamomyces is suppressed. Therefore, in the present invention, the growth of lactic acid bacteria is suppressed by maintaining anaerobic conditions, and the incomplete bacteria belonging to the genus Wickerhamomyces are controlled so as to be advantageously maintained.
With the intention of confirming this, a result of culturing under aerobic conditions is also shown as a comparative experiment. Here, the aerobic condition specifically refers to culturing a medium inoculated with imperfect bacteria belonging to the genus Wickerhamomyces in a state of being substantially aerated with the outside air.

[Results of solid-phase fermentation in demonstration experiments]
Culture tests and biogas generation experiments were conducted at the Hyogo Prefectural Industrial Technology Center, which is a public testing and research institution.
FIG. 5 shows the result of biogas recovery in the demonstration experiment.
FIG. 5 shows the results of GC-MS measurement of biogas released around the third day from the start of culture.
The type of gas was identified by GC-MS (Gas Chromatography Mass Spectrometry).

GC-MS measurement results were analyzed.
In FIG. 5, the peak of 2.53 minutes clearly detected is methanol, and the peak of 2.92 minutes is ethanol. These are considered to be biogas generated in the flask for solid phase fermentation assuming the biogas generation unit 110. Others are not considered to be biogas. For example, a large peak near 2 minutes and a peak at 8.5 minutes are considered to be acetone-derived peaks used as a solvent for the trap solution. The two peaks at 18.04 are presumed to originate from the material contaminated with the plasticizer peak of the plastic. Many other small peaks are considered non-reproducible noise.

Next, the time series change of the kind of generated biogas observed through the culture days was examined.
FIG. 6 is a diagram showing a time-series change in the type of generated biogas observed through the number of days of culture. The culture days are plotted on the horizontal axis, and the biogas production concentration (mg / l) is plotted on the vertical axis.
As shown in FIG. 6, while the days of culture were shallow, alcohols such as methanol and ethanol were actively generated and the alcohol concentration was high. However, as the days of culture passed, the ester concentration increased. You can see that That is, it can be read that the alcohol concentration is first improved by the culture, and the ester concentration is gradually improved so as to be delayed. That is, it can be seen that ethanol fermentation first proceeds, and ester fermentation subsequently occurs.

When the biogas component released into the air was examined by GC-MS, it was found that the main component of the alcohols released into the air was ethanol and the esters were ethyl acetate.
From this experimental result, by the biogas production method of the present invention used in the experimental apparatus assuming the biogas generation apparatus of the present invention, the incomplete bacteria belonging to the genus Wickerhamomyces, under solid-state fermentation conditions such as anaerobic state It was proved that biogas can be obtained from the carbon source of bamboo powder.

Here, it was confirmed that the biogas production method of the present invention enabled solid-phase fermentation.
FIG. 7A shows a state near the bottom surface of the flask for solid phase fermentation assuming the biogas generation unit 110 immediately after the end of the experiment. As shown to Fig.7 (a), the inside of the flask for solid phase fermentation remained a solid phase, and the liquid pool was not seen. Even if the bamboo powder inside the solid phase fermentation flask was directly confirmed, it was not possible to confirm the state of being wet with liquid. Methanol and ethanol captured by the cool trap reach the cool trap device assuming the biogas recovery device 130 in a gas phase state, so that gas phase biogas is generated from the solid phase fermentation without going through the liquid phase. It can be seen that solid-phase fermentation progressed in the cycle.

Here, the life cycle of imperfect bacteria belonging to the genus Wickerhamomyces at the time when the production of biogas in the second step was completed was confirmed.
The life cycle was confirmed by culture using a medium.
For the adjustment of the extract, 25 g of the sample was dispersed and left in 500 ml of sterilized water, and the supernatant was collected and extracted.
As the type of medium, potato dextrose medium and YPD medium were used. The potato dextrose medium is suitable for the propagation of incomplete bacteria belonging to the genus Wickerhamomyces in the filamentous fungus-like life cycle. It can be estimated that it was a life cycle. The YPD medium is suitable for the propagation of bacteria in the yeast-like life cycle, and if the YPD medium is highly propagated, it can be estimated that the life cycle of the bacteria was a yeast-like life cycle.
In any case, the culture conditions were such that 200 μL of the extract was seeded on the surface of the medium, sealed with a sealing tape, and then allowed to stand at 29 ° C. for 3 days.

The left figure of FIG.7 (b) is the culture result using a potato dextrin culture medium. It can be seen that the growth of imperfect bacteria belonging to the genus Wickerhamomyces sowed is relatively limited, although it can be seen.
The right figure of FIG.7 (b) is a culture result using a YPD culture medium. It can be seen that the inoculated bacteria belonging to the genus Wickerhamomyces sowed have large growth and are actively breeding.
From the above, it was confirmed that the life cycle of the incomplete bacteria belonging to the genus Wickerhamomyces at the time when the production of the biogas in the second step was completed was changed to a yeast-like life cycle.

[Results and Discussion]
Consider the results of the above demonstration experiment.
It was confirmed that the biogas generation process is solid-phase fermentation in which biogas is generated in a gas phase from a solid-state carbon source and biogas is generated directly from the carbon source without going through the liquid phase. Therefore, it is considered that the biogas production method of the present invention produced alcohols, volatile organic acids, and esters thereof in a solid phase fermentation cycle in the process of propagation of imperfect bacteria belonging to the genus Wickerhamomyces.

The solid-phase fermentation cycle using imperfect bacteria belonging to the genus Wickerhamomyces is performed by transitioning between different life cycles of the filamentous fungi-like life cycle and the yeast-like life cycle.
The first reaction is the reaction that occurs at the beginning of the solid-phase fermentation cycle. The carbon source is in a cellulose-rich and glucose-deficient state, such as bamboo flour. By placing it under anaerobic conditions, the incomplete bacteria belonging to the genus Wickerhamomyces Cellulose is decomposed in a filamentous fungus-like basidiomycetous life cycle to produce glucose.
In the production of glucose, it is considered that first, incomplete bacteria belonging to the genus Wickerhamomyces in the “Basidiomycetous fungus-like life cycle” undergo a delignification reaction that decomposes cellulose and lignin. This can be verified by measuring the decrease in lignin in a demonstration experiment.
Next, following the delignification reaction, incomplete bacteria belonging to the genus Wickerhamomyces in the “Basidiomycetous basidiomycetous life cycle” are considered to cause a glucose decomposition reaction that decomposes cellulose into glucose.

In the second reaction, when the glucose decomposition reaction proceeds in the first reaction and the glucose concentration in the surrounding environment increases, the imperfect bacteria belonging to the genus Wickerhamomyces gradually change their life cycle to “Family fungus basidiomycetous life cycle”. To “Yeast-like life cycle”. In other words, by placing the carbon source under glucose-rich conditions, the life cycle of imperfect bacteria belonging to the genus Wickerhamomyces is transitioned to the “yeast-like life cycle” and activated, and glucose is decomposed into alcohols (ethanol). Furthermore, the production of esters (ethyl acetate) is promoted.

Both the first reaction and the second reaction described above are performed in a solid phase and never go through a liquid phase. Since the released alcohols and esters are released as a gas phase, it has been verified that a gas phase biogas is generated from a solid state carbon source.

In Example 2, the first step and the second step are alternately and continuously repeated to produce a biogas in a gas phase directly from a solid phase carbon source without passing through a liquid phase. It is a manufacturing method.
FIG. 8 is a diagram simply illustrating the biogas production apparatus of the present invention according to Example 2 and the biogas production method of the present invention performed in the apparatus.
Compared to FIG. 2, an oxygen supply means 150 and a valve 151 are added. The oxygen supply means 150 may be an industrial oxygen cylinder or the like. However, since the aerobic condition in the present invention may be the same oxygen concentration as that of air in the atmosphere, a pump for sending air in the atmosphere or a ventilation of the atmosphere. It can be ventilation.
In the biogas production method of Example 2, after the biogas is recovered in the first cycle through the first step from the first step, the transition from the second step to the first step is continued. Then perform the next biogas production cycle.
FIG. 8B shows a state in which the first step and the second step are repeated. By repeating this first step and the second step alternately and continuously, it is a step of continuously producing biogas in the gas phase directly from the carbon source in the solid phase without going through the liquid phase. Yes.
In this example, the conditions in the first step are different from the conditions in the first step after the first time.

Also in the case of the second embodiment, the method for generating the biogas by passing through the first first step to the second step is the same as that of the first embodiment, and thus the description thereof is omitted here.
In the case of Example 2, the process continues from the second process to the first process again.
FIG. 9 simply shows that the first step goes through the second step and then returns to the first step. As shown in FIG. 9, the culture condition setting means 130 closes the valve 141 of the nitrogen supply means 140 and opens the valve 151 of the oxygen supply means 150 to supply oxygen into the culture tank 110. Is an aerobic condition. In an industrial mass production system, the oxygen supply means 150 may be a pump that sends out air in the atmosphere, a ventilation that ventilates the atmosphere, an industrial oxygen cylinder, or the like.

A demonstration experiment for demonstrating the procedure of returning from the second step to the first step was performed by replacing the air in the experimental instrument configuration shown in FIG. 3 with air. Other laboratory equipment configurations were left as they were.
The solid phase fermentation flask in which air was replaced was continued to be cultured in a 30 ° C. incubator for 1 week.
FIG. 9B shows the life cycle of imperfect bacteria belonging to the genus Wickerhamomyces obtained after one week of culture. The confirmation of the life cycle was confirmed by culture using a medium as in the previous confirmation.
For the adjustment of the extract, 25 g of the sample was dispersed and left in 500 ml of sterilized water, and the supernatant was collected and extracted. As the type of medium, potato dextrose medium and YPD medium were used. In any case, the culture conditions were such that 200 μL of the extract was seeded on the surface of the medium, sealed with a sealing tape, and then allowed to stand at 29 ° C. for 3 days.

The left figure of FIG.9 (b) is the culture result using a potato dextrin culture medium. It can be seen that the inoculated bacteria belonging to the genus Wickerhamomyces sowed have large growth and are actively breeding.
The right figure of FIG.9 (b) is the culture result using a YPD culture medium. It can be seen that the inoculated bacteria belonging to the genus Wickerhamomyces sowed have large growth and are actively breeding.
From the above, by making the aerobic condition after the second step, some imperfect bacteria belonging to the genus Wickerhamomyces re-transitioned from the yeast-like life cycle to the filamentous basidiomycetous life cycle. I was able to confirm. In other words, it was confirmed that the process could return from the second process to the first process.

In this demonstration experiment, it seems that a part of the life cycle of imperfect bacteria belonging to the genus Wickerhamomyces was maintained as a yeast-like life cycle from the experiment on the right side of FIG. 9B. It can be expected that the life cycle of imperfect bacteria belonging to the genus Wickerhamomyces re-transitions to the filamentous fungus-like life cycle if the culture period is lengthened. Moreover, even if the life cycle of some incomplete bacteria belonging to the genus Wickerhamomyces continues to maintain the yeast-like life cycle, it does not significantly inhibit the decomposition of cellulose into glucose.
As described above, it was confirmed that after the second step, it was possible to return to the first step and transition again to the step of decomposing cellulose into glucose.

Next, in Example 2, after returning to the 1st process, it shifts to the 2nd process again.
FIG. 10 simply shows the transition from the state returned to the first step to the second step as shown in FIG. As shown in FIG. 10 (a), the culture condition setting means 130 closes the valve 151 of the oxygen supply means 150 and opens the valve 141 of the nitrogen supply means 140 to supply nitrogen into the culture tank 110. The inside of 110 is an anaerobic condition. In the demonstration experiment, the inside of the fermentation flask was replaced with nitrogen from a nitrogen cylinder in the experimental instrument configuration shown in FIG. Other laboratory instrument configurations can be left as they are.
This experiment was the same as the previous one, and it was confirmed that biogas can be generated and recovered.

Thereafter, as shown in FIG. 10B, the first step and the second step may be repeated.
Here, the advantage which repeats a 1st process and a 2nd process alternately is demonstrated.
In general, fermentation technology for obtaining bioethanol using a carbon source is conducted by other methods, such as hydrolyzing the carbon source and physically adding a large amount of energy. Since most of them are changed, there is no concept of repeating the fermentation process as in the present invention. However, in the present invention, the two steps of carbon source cellulose to glucose and glucose to ethanol are performed only with imperfections belonging to the genus Wickerhamomyces.

Here, it is considered that it takes a long time to ferment the cellulose, which is a carbon source, until it becomes almost glucose with only imperfections belonging to the genus Wickerhamomyces. Then, in order to advance fermentation progress efficiently, the method of advancing alternately two processes called glucose from a carbon source, glucose, and glucose to ethanol was recalled.

FIG. 11 is a diagram simply showing changes in the carbon source. An example of the carbon source of the present invention is a powder such as bamboo powder, and a large surface area is ensured. However, when enlarged, it is a lump of cellulose as shown in FIG. Here, as shown in the upper to middle of FIG. 11, when an incomplete bacterium belonging to the genus Wickerhamomyces decomposes cellulose in the life cycle of a filamentous fungus-like fungus and changes it to glucose, the glucose covers the surface. Since the present invention is premised on solid phase fermentation, it is considered that glucose does not elute and covers the surface. Even if the culture is continued in a state where the life cycle of the incomplete bacterium belonging to the genus Wickerhamomyces is maintained as a filamentous fungus-like life cycle, the activity may be reduced. Therefore, in the present invention, the life cycle of an incomplete bacterium belonging to the genus Wickerhamomyces is transitioned to a yeast-like life cycle that actively degrades glucose. As shown in the middle to the bottom of FIG. It is fermented and evaporated to recover as biogas first. By fermenting the glucose in the solid phase on the surface at an appropriate place as described above, the cellulose is exposed again near the surface. Therefore, the process returns again from the lower stage to the upper stage in FIG. 11, and the incomplete bacteria belonging to the genus Wickerhamomyces are returned. The life cycle is returned to the filamentous fungus-like basidiomycetous life cycle, and the fermentation process of cellulose to glucose is performed. Thus, the activity efficiency of the fermentation as a whole is increased by repeating the first step and the second step.

The preferred embodiments of the biogas production method and biogas production apparatus of the present invention have been illustrated and described above, but it will be understood that various modifications can be made without departing from the technical scope of the present invention. Will.

The biogas production method of the present invention can be widely applied to technical fields involving biomass processing using plant biomass resources, for example, lignocellulosic biomass resources such as trees, vegetation, bamboo, and rice straw.

DESCRIPTION OF SYMBOLS 100 Biogas production apparatus 110 Culture tank 120 Carbon source input means 130 Culture condition setting means 140 Nitrogen supply means 141 Valve 150 Oxygen supply means 151 Valve 160 Biogas recovery part

Claims

A biogas production method comprising a biogas production step of producing biogas by culturing an incomplete bacterium belonging to the genus Wickerhamomyces together with a carbon source,
The imperfect bacteria belonging to the genus Wickerhamomyces are those that transition through a different life cycle of a filamentous fungus basidiomycetous life cycle and a yeast-like life cycle,
The biogas generation step includes
A first step of culturing an incomplete bacterium belonging to the genus Wickerhamomyces in a filamentous fungus basidiomycetous life cycle and decomposing the carbon of the carbon source in a solid state into glucose;
Incomplete bacteria belonging to the genus Wickerhamomyces are cultured in a yeast-like life cycle, and the solid-phase glucose produced from the solid-state carbon source in the first step is decomposed to produce vapor-phase methanol. A second step of producing biogas which is ethanol, one of their esters or a mixture thereof,
In the first step and the second step, the carbon source is transferred without passing through a liquid phase while transitioning the filamentous fungi-like basidiomycetous life cycle and the yeast-like life cycle of the incomplete bacterium belonging to the genus Wickerhamomyces. A method for producing a biogas, which is a process for generating the biogas in a gas phase directly from a gas phase.
The biogas in a gas phase state is directly produced from the carbon source in the solid phase state without passing through a liquid phase by continuously and alternately repeating the first step and the second step. The biogas production method according to claim 1.
The condition for transition from the first step to the second step is a yeast-like life cycle activity condition for activating the yeast-like life cycle of an incomplete bacterium belonging to the genus Wickerhamomyces,
The condition for transition from the second step to the first step is a filamentous fungus basidiomycetous life cycle activity condition for activating the filamentous fungi basidiomycetous life cycle of the incomplete bacterium belonging to the genus Wickerhamomyces,
While appropriately switching between the yeast-like life cycle activity condition and the filamentous basidiomycetous life cycle activity condition as the culture conditions of the culture tank, the first step and the second step are alternately switched. The biogas production apparatus according to claim 2, wherein the biogas production is advanced.
The yeast-like life cycle activity conditions are normal temperature and anaerobic conditions,
The method for producing biogas according to claim 3, wherein the filamentous basidiomycetous life cycle activity condition is normal temperature and aerobic condition.
In alternating repetition of the first step and the second step,
In the first step, the culture conditions are not the filamentous basidiomycetous life cycle activity conditions, but are initially set to room temperature and anaerobic conditions as the yeast fungus life cycle activity conditions, and the Wickerhamomyces genus The life cycle of an incomplete bacterium belonging to is controlled by the filamentous fungus basidiomycetous life cycle to suppress the growth of lactic acid bacteria mixed in the carbon source, and the incomplete bacterium belonging to the genus Wickerhamomyces While the cellulose of the carbon source is decomposed into glucose, the life cycle naturally transitions from the filamentous basidiomycetous life cycle to the yeast-like life cycle under the yeast-like life cycle activity conditions. 5. The biogas production method according to claim 3, wherein control is performed so as to make a transition to the second step.
In alternating repetition of the first step and the second step,
Excluding the transition from the first step to the second step,
Switching from the first step to the second step is an arbitrary timing after the decomposition rate of cellulose from the surface of the carbon source to glucose has passed a peak,
The switching from the second step to the first step is performed at an arbitrary timing after the decomposition rate of the glucose into the biogas which is the ethanol, their esters, or a mixture thereof passes the peak. The biogas production method according to any one of claims 2 to 5, wherein the biogas production method is provided.
The method for producing biogas according to any one of claims 1 to 6, wherein the carbon source is a plant biomass material.