JP5856811B2 - Hydrogen gas production method using apple - Google Patents

Description

  The present invention relates to a technology for obtaining hydrogen by fermenting non-pathogenic bacteria using biomass such as apple pomace, apple juice, or a mixture thereof as raw materials.

  It is said that the consumption of fossil fuels such as oil increases the amount of carbon dioxide in the atmosphere and causes global warming. Thereby, since the carbon neutral effect is obtained, a method of using biomass as an alternative energy source for oil has attracted attention.

  In particular, hydrogen derived from biomass can be used as a reaction gas for a fuel cell, and since it is only water that is generated at the time of power generation, it has been attracting attention as a clean energy material.

  Hydrogen production from biomass can be performed by microorganisms that perform hydrogen fermentation. Examples of microorganisms used for hydrogen production include anaerobic bacteria such as Clostridium, Enterobacter, and Escherichia. However, most of these bacteria have pathogenicity with a biosafety level of 2, and most of them require attention to safety during the fermentation process.

  It is also known that photosynthetic bacteria also perform hydrogen fermentation, but fermentation requires a supply of light energy, and when sunlight is used, there is a problem that fermentation is efficient only on sunny days. is there. Cyanobacteria and chlorella are also known to produce hydrogen, but the hydrogen generation rate is slow and it is said that it is difficult to put into practical use.

  There are various types of biomass, but as one of the unused biomass, it is also important to use apple pomace (processed residue) during juice production and apples that are agricultural waste due to non-standard or unsold items It is said that. However, there are few reports on its use as biomass energy, although there are some uses as feed straw.

Electron, ion and functional chemistry series, Vol. 4, “All about polymer electrolyte fuel cells”, Section 4, Biomass hydrogen production, p234, Supervisor Hideo Tamura, Author Author Shigeharu Tanio, October 1, 2003, first edition, published by NTS Corporation

  The present invention was created in order to solve the above-mentioned disadvantages of the prior art, and its purpose is not to use pathogenic microorganisms, it is not necessary to use fermentation starters such as inoculum or activated sludge, and from biomass apples. A method capable of producing biohydrogen is provided.

In order to solve the above-mentioned problems, the present invention contains either or both of the squeezed straw and the apple juice out of the squeezed straw and the apple juice when the apple fruit is squeezed, and the pH is 6.5 or more. 8 to prepare a culture adjusted to less than is the hydrogen gas producing method for releasing hydrogen gas by fermenting the broth with no pathogenic Rhodosporidium Toruroido (Rhodosporidium toruloides) adhering to the apple fruit .
Also, the present invention is a hydrogen gas producing method of Ru fermented by maintaining the culture in 30 ° C. or higher.

Hydrogen gas is obtained from unsold apple fruits and apple agricultural waste such as apple fruit squeezed straw.
Since fermentation is carried out using yeast belonging to the genus Rhodosporidium, which is a resident bacteria of apple fruits, there is no need for a step of administering bacteria from the outside, such as activated sludge and inoculum.
When fermented at pH 7 or higher, or near pH 7, which is optimal for the genus Rhodosporidium, hydrogen gas collection is high.

An example of a hydrogen collecting apparatus capable of carrying out the hydrogen gas production method of the present invention A graph showing the relationship between the number of days of cultivation and the integrated value of gas generation by the type of apple broth Graph showing the relationship between the integrated value of light and gas generation amount Graph showing the relationship between pH and integrated value of gas generation Graph showing the relationship between sterilization and fermentation Graph showing the relationship between the number of days of cultivation of grapes and peaches and the integrated value of gas generation Graph showing the relationship between the pH of San Tsugaru (from Nagano) and the amount of gas generated Graph showing hydrogen gas generation status at high pH Graph showing an example when hydrogen gas is generated at low pH

  Natural apples have yeast, Rhodosporidium toruloides, which adheres to the fruit of the apple. Since apples are biomass, they are not subjected to disinfection, heating, sterilization, or sterilization. Then, hydrogen fermentation is performed using the resident bacteria of the natural apple.

The culture solution consisting of apple pomace and apple juice, or a mixture thereof, usually has a pH in the range of about 4-5. By adjusting this pH to around 7, it is possible for yeasts of the genus Rhodosporidium to undergo hydrogen fermentation. Use as an environment for fermentation.
In the present invention, the apple pomace contains a portion of the apple skin, and the apple juice (apple juice) is obtained by squeezing the pulverized material containing the apple fruit skin.

  Apple pomace and apple juice, or a mixture thereof are put in a container together with water (hereinafter referred to as fermentation solution). The apple pomace and apple juice, or a mixture thereof contains Rhodosporidium toruloides, which are resident bacteria, and there is no need to add a fermentation starter such as an inoculum or activated sludge. The pH of the fermentation solution is about 4 to 5 when not adjusted.

Since hydrogen fermentation is not performed under these conditions, the fermentation solution is left to stand for about 24 to 200 hours while maintaining a temperature of about 30 ° C. while adjusting the pH to around pH 7 where Rhodosporidium toruloides performs hydrogen fermentation. Gas is generated from The generated gas contains hydrogen and carbon dioxide, and the hydrogen gas (H ₂ ) concentration is about 30 to 80%. This hydrogen concentration is a concentration sufficient to drive the fuel cell, and power generation using the gas obtained from the fermentation solution, that is, utilization of biomass energy becomes possible. When performing the fermentation, from the viewpoint of the hydrogen collection rate, it is desirable to maintain the temperature of the fermentation broth in the range of 10 ° C. or higher and 40 ° C. or lower. desirable.

In addition, the concentration of the culture solution can be adjusted to a desired value for fermentation, but the culture solution in which apple juice is not diluted from the culture solution (0.3%) in which 1 g of squeezed rice cake is dispersed and dissolved in 330 mL of water It is possible to produce hydrogen by fermenting a culture solution of any concentration up to (100%).
However, in view of efficiency, a culture solution having a concentration of 3% or more obtained by dissolving 10 g of pomace in 300 mL of water is desirable.

For hydrogen storage, for example, the hydrogen collection device 10 described in FIG. 1 can be used.
This hydrogen collection device 10 has a fermenter 12 and a collection tank 22, and the fermenter 12 is closed with a lid 13 at a ceiling.
One end of a collection pipe 32 is connected to the lid 13, the other end of the collection pipe 32 is introduced into the collection tank 22, and the opening 18 at the tip of the other end is an abbreviation of the bottom surface of the collection tank 22. It is arranged above the center.
When the pH-adjusted culture solution 15 is disposed in the fermenter 12 and fermented to generate hydrogen, the fermenter 12 is discharged through the collection pipe 32 into the collection tank 22 through the collection pipe 32.

  Above the opening 18, a storage tower 23 having a lower end disposed below the upper end of the collection tank 22 is disposed. The lower end of the storage tower 23 is opened and formed into a container shape, and the collected water 16 is poured into the collection tank 22 in advance, and the lower end of the storage tower 23 is immersed in the water leveling position of the collected collected water 16. If this is done, the hydrogen released from the opening 18 rises as bubbles in the collected water 16 and is collected in the storage tower 23 by water replacement. When hydrogen is collected, the storage tower 23 rises due to the buoyancy generated according to the volume.

One end of the sample tube 34 is connected to the bottom surface directed upward of the storage tower 23 so that the hydrogen stored in the storage tower 23 flows through the sample tube 34 and is introduced into the fuel cell 26. It has become.
A cock 33 is provided in the middle of the sample tube 34. When the hydrogen released from the opening 18 is stored in the storage tower 23, the cock 33 is closed and the fuel cell 26 is stored with the stored hydrogen. When operating, the cock 33 is opened, and the pressure at which the storage tower 23 descends due to gravity is applied to the hydrogen inside the storage tower 23, and hydrogen is introduced into the fuel cell 26 at that pressure.

  A fan motor 27 is connected to the fuel electrode 36 and the oxygen electrode 37 of the fuel cell 26. When the hydrogen is supplied to the fuel cell 26 to generate electric power and a voltage is applied to the fan motor 27, the fan motor 27. The propeller connected to is designed to rotate.

  Therefore, since the rotation of the propeller allows visual recognition of the results from the generation of hydrogen by fermentation in the fermenter 12 through the collection by the storage tower 23 to the generation of power by the fuel cell 26, the hydrogen collection device 10 Can be used as teaching materials for reusing apple waste, and if the hydrogen collecting device 10 is enlarged, it can also contribute to diversification of energy by waste recycling.

  One apple was lightly washed with water, then cut into four with a knife, and then grated with a food grinder. The apple that was grated was squeezed out in a 300 mL glass beaker using a plastic net (draining net for kitchens), and the pomace was subjected to the test. In addition, the glove work was carried out with rubber gloves in order to prevent contamination from resident bacteria.

10 g of the prepared pomace was placed in a 300 mL glass Erlenmeyer flask and 330 mL of deionized water was added. To this, 0.28 g of KH ₂ PO ₄ , 0.24 g of K ₂ HPO ₄ , 0.49 g of HNaCO ₃ (ratio is 0.85 g of KH ₂ PO ₄ , 0.75 g of K ₂ HPO ₄ , 1.5 g of HNaCO ₃ To 1 L), and a culture solution having a pH adjusted to around 7 was prepared (by just adding the reagent, the pH was around 7). The pH of the liquid before and after the culture was measured with a pH meter (Horiba, Ltd. “B-212”).

  A conical flask containing a Teflon tube (Teflon is a registered trademark) was inserted into the Erlenmeyer flask containing this solution, and the Erlenmeyer flask was heated to a temperature of around 30 ° C. (measured temperature: 31 ° C.) by illumination, and an adjusted thermostat ( The flask was placed in a water tank (without using water), and the microorganisms in the culture were cultured and fermented. The tip of a Teflon tube (Teflon is a registered trademark) was submerged in a water tank containing water, and the gas generated from the tip of the tube was replaced with water in a glass graduated cylinder to collect the generated gas.

The relationship between the number of days of culture and the integrated value of gas generation is shown in FIG. The culture solution using the Sanfuji apple and the culture solution using the Fuji apple were separately fermented without sterilization to collect the generated gas.
From the culture solution, generation of gas could be confirmed after 1-2 days.

  The gas collected in the graduated cylinder is collected with a plastic syringe and then diluted 100-fold with air with a glass syringe, and then the hydrogen concentration is detected by the detector tube method (hydrogen detector tube U-type Komyo Chemical Co., Ltd.) The hydrogen concentration was measured. The measured hydrogen concentration in the generated gas was 50% when converted to the concentration before dilution.

Furthermore, 10 mL of the generated gas recovered by the syringe was put into the fuel cell, and it was confirmed whether or not the connected fan motor was driven. The driving time was 6 minutes 2 seconds for Sanfuji and 7 minutes 11 seconds for Fuji.
By introducing the generated gas into the fuel cell, power generation was confirmed from the drive of the fan. From this, it was confirmed that electric energy could be extracted from the apple which is biomass.
In addition, although there are two hydrogen inlets of the fuel cell, a syringe was separately connected to the inlet that is not used for charging so that the charged hydrogen would not diffuse from the fuel cell.

On the other hand, a culture broth was also prepared from apples sterilized (high-pressure steam sterilization) as a comparative control and fermentation was attempted under the same conditions, but no gas was generated. From this, it was confirmed that the microorganisms contained in the apple fermented with hydrogen and produced hydrogen.
It was also found that when apple biomass was subjected to sterilization, sterilization, sterilization, etc., such as heating, resident bacteria were killed, so that fermentation was not performed and hydrogen production was not observed.

<Optical test>
Using a Fuji apple juice having a concentration of 30%, the pH was adjusted to 6.5 to prepare a culture solution. The amount of gas generated in this culture solution was measured when the fermentation vessel in which the culture solution was arranged was shielded from light and when fermented while being irradiated with light. The culture solution was maintained at 30 ° C. for fermentation.

The relationship between the number of days of culture and the integrated value of gas generation is shown in FIG. Gas is generated regardless of the presence or absence of light irradiation. This result indicates that hydrogen production is not caused by photosynthetic bacteria. The hydrogen concentration of the generated gas was 30% when irradiated with light and 25% when not irradiated with light.
When 10 mL of the generated gas was extracted with a syringe and injected into the fuel cell and operated, the rotation time of the fan motor was 10 minutes or more when the generated gas was irradiated with light and when the light was not irradiated. .

<PH test (1)>
Next, 10 g of Fuji squeezed straw was put into 330 mL of deionized water to prepare culture solutions adjusted to pH 4.9, 7.0, and 7.2, respectively, and each culture solution was fermented at 30 ° C. I let you. pH 4.9 is not adjusted for pH.

FIG. 4 shows the relationship between the initial pH value (at the start of fermentation) and the integrated value of the amount of gas generated. Gas generation was not confirmed at pH 4.9, but gas was generated around pH 7 (pH adjustment), and the hydrogen concentration was 40% in the culture solution at pH 7.0, and 45 in the culture solution at pH 7.2. %Met. This shows that apple biomass does not produce hydrogen when pH is not adjusted, but can produce hydrogen by adjusting the pH to a neutral range.
When 5 mL of the obtained generated gas was extracted with a syringe and injected into the fuel cell to operate, the fan motor rotated for 10 minutes or more.

<Identification of fermentation microorganisms>
A portion of a gas-producing fermentation solution (48 hours after fermentation) was collected, and this was agar-agar medium (agar plate medium prepared by sterilization after adjusting apple juice to pH 6.7. Agar concentration was 1.5). %)) And tried to catch microorganisms in the fermentation solution. As a result, a large number of red colonies and a small amount of white colonies were confirmed on the agar medium 72 hours after smearing.

The number of red bacteria in the fermentation solution was about 5 × 10 ⁴ / mL, and the number of white bacteria was about 2 × 10 ² / mL. Since most of the colonies that grew were red fungi, the microorganisms contributing to hydrogen production were considered red fungi, and an attempt was made to identify these red fungi.

  The DNA of S. cerevisiae was extracted and the DNA of the large subunit rRNA D2 region was amplified by PCR. The base sequence of the amplified DNA was analyzed using ABI PRISM 310 Genetic Analyzer (Life technologies corporation). Homology search of the obtained sequence with sequences registered in the international nucleotide sequence database (DDBJ / EMBL / Genbank) and the database of Microseq ID Analysis Software Version 2.0 (Life technologies corporation), and a phylogenetic tree with related species Was created by the neighbor join method (NJ method).

  As a result, the homology was highest with Rhodosporidium toruloides, and the homology was 99.44%. From this result, it was found that the red fungus, which is the main microorganism in the fermentation solution, is Rhodosporidium toruloides. Rhodosporidium toruloides is isolated from plants, soil, air, animals, seawater, etc., and its incomplete era is Rhodotorula rubescens.

Three platinum loops of Rhodosporidium toruloides colonies were added to a culture solution of 30% of sterilized apple juice and pH 6.7, and cultured at 30 ° C. As shown in FIG. 5, gas was generated and hydrogen was also confirmed. . The hydrogen gas concentration was 30%. (In the graph of FIG. 5, data is not acquired during the 3rd to 8th days from the start of fermentation.)
When 5 mL of the generated gas was extracted with a syringe and injected into the fuel cell and operated, the rotation time of the fan motor was 2 minutes 30 seconds.
Hydrogen was not generated in comparison subjects cultured at 30 ° C. without introducing colonies into the same culture solution.

From the above results, it was confirmed that Rhodosporidium toruloides contributed to hydrogen generation from apple biomass.
Rhodosporidium toruloides has a biosafety level of 1 and is not pathogenic. From this, it can be said that the production of biohydrogen using this microorganism is highly safe during fermentation, hydrogen production and post-treatment operations.

<Analysis of evolved gas>
The generated gas was analyzed by the detector tube method. An example of the results is shown in Table 1 below. All the detector tubes are manufactured by Komyo Chemical Co., Ltd.

The results in Table 1 show examples of measurement results when measured multiple times. The timing of measurement and the test lot of apples used are not the same.
However, it is understood that hydrogen and carbon dioxide are the main generated gases, and hydrogen sulfide, acetic acid, ethylene, alcohols, and ammonia are hardly contained.

  Since the generated gas does not contain hydrogen sulfide, which is a catalyst poisoning substance of the fuel cell, there is an advantage as an input gas to the fuel cell. Since no harmful gas such as ammonia is contained, there is a high safety advantage.

<Concentration of sugar, organic acid and ethanol in fermentation solution before and after fermentation>
The sugar and organic acid concentrations in the fermentation solution before and after fermentation were measured.
The measurement results are shown in Table 2 below.

At the time of this test, 65 mL of gas containing 40% hydrogen was produced.
Each component was measured by an enzyme method (Roche F-kit). As a result, it was confirmed that sucrose, maltose, glucose, fructose saccharides and malic acid were contained at a relatively high concentration before fermentation, but significantly decreased after fermentation. On the other hand, ethanol, L / D-lactic acid, and acetic acid were hardly contained before fermentation, but it was confirmed that they were remarkably increased after fermentation. From this result, it can be seen that microorganisms use carbohydrates and malic acid as nutrient sources in the fermentation solution, and thus hydrogen fermentation, ethanol fermentation, lactic acid fermentation, and acetic acid fermentation are performed.
The pH was 7.1 before fermentation, but decreased to 5.8 after fermentation. This shows that organic acids, such as acetic acid and lactic acid, produced | generated by fermentation.

<Hydrogen production rate>
The hydrogen production rate in the present invention depends on the test conditions, but as an example, it is 22 mmol / L. The result of about h is obtained. According to the literature, the production rate of non-pathogenic hydrogen-producing microorganisms is 0.4 for Oscillatoria sp. (Joremo), 1.2 for Anabaena cylindrical (Cyanobacteria), 5.3 mmol / Rhdopseudomonas capslata (Photosynthetic bacteria). L. h. This shows that the hydrogen production rate according to the present invention is excellent as a non-pathogenic microorganism. Among pathogenic microorganisms, Clostridium beijerinkii (obligate anaerobe) is 17 and Enterobacter aerogenes is 36 mmol / L. Although h, there are cases earlier than the present invention, it can be seen that the method of the present invention is sufficiently non-pathogenic but comparable to the production rate of these pathogenic microorganisms.

<Comparative example>
Instead of natural apples, grapes and peaches were also fermented by resident bacteria attached to the fruits. The result is shown in FIG.
Although gas was generated for peaches and grapes, 75% hydrogen gas concentration was detected in the peach gas, but no hydrogen was detected in the grape gas.
For grapes, 40 g of squeezed koji for Kyoho is added to 330 mL of deionized water, and for peaches, 20 g of squeezed koji of white peach is added to 330 mL of deionized water, and pH is set to 8.0 to prepare a culture solution Fermented at 30 ° C.

When 5 mL of peach and grape gas was extracted with a syringe and injected into the fuel cell, the operation time of the fan motor was 10 minutes or more with peach gas, and it did not rotate with grape gas. It was.
In addition, at the time of confirming hydrogen generation, the resident bacteria that contributed to fermentation have not been identified.

<PH test (2)>
The production and type of apples were changed, and hydrogen generation was tested.
The test results are shown in Table 3 below.

From Table 3 above, it can be seen that hydrogen generation is possible when the pH value at the start of fermentation is 6 pH or more and less than 8 pH.
Next, 10 g of apples of “Sun Tsugaru” produced in Nagano Prefecture are dissolved in 300 mL of water, and the pH value is 4.9, 5.8, 7.0, 7.9 at a concentration (3%). Were prepared and fermented. The culture temperature was 30 ° C.

  FIG. 7 shows the relationship between the number of culture days and the gas generation amount integrated value for each culture solution. The numerical values in the figure are values indicating the hydrogen gas concentration in the generated gas and the motor rotation time by the sample gas for each culture solution (injection of 5 mL with a syringe). From these test results, hydrogen generation was confirmed in the culture solution having a pH of 5 or more and less than pH 8.

Next, instead of Nagano products, Aizu “Sun Tsugaru” apples with different pH values of 5.8, 7.0, 8.0, 9.1 in the same concentration and volume as Nagano products were prepared. Fermented under the same conditions as in Nagano.
FIG. 8 shows the relationship between the number of days of culture and the integrated value of the amount of gas generated. The injection amount into the syringe is 5 mL. Hydrogen gas generation was confirmed in the culture solution of pH 7.0, but hydrogen gas generation was not confirmed in the culture solutions of pH 5.8 and pH 8-9.

Next, an example in which a large amount of hydrogen gas is generated even at a low pH value will be described.
Fig. 9 shows the number of days of cultivation and the amount of gas generated when fermenting the fermented juice of 30% of Aomori apple "Fuji", pH 6.5, and fermenting it with light. It is a graph which shows the relationship with the integrated value of. The hydrogen gas concentration when irradiated with light was 30%, and the hydrogen gas concentration when shielded from light was 25%. The motor driving time when 10 mL was injected with a syringe was 10 minutes or more, respectively.
As can be seen from the example of this graph, even when the pH is 6.5, a large amount of hydrogen gas may be generated.
From the above, for hydrogen gas generation, the pH value of the culture solution is preferably 6 or more and less than 8, and considering the generation amount, it is preferably 6.5 or more and less than 8.0.

DESCRIPTION OF SYMBOLS 10 ... Hydrogen collection device 12 ... Fermenter 23 ... Storage tower 26 ... Fuel cell 27 ... Fan motor

Claims (2)

Of lees and apple juice when squeezed apple fruit, it contains either one or both of the lees or the apple juice, to produce a pH was adjusted to 8 less than 6.5 or more cultures A method for producing hydrogen gas, wherein the culture solution is fermented by Rhodosporidium toruloides having no pathogenicity attached to the apple fruit to release hydrogen gas. Claim 1 of hydrogen gas production method according to Ru fermented by maintaining the culture in 30 ° C. or higher.

Images