JP2010088334A - Method for producing starch and ethanol by using algae releasing starch to exterior of cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing bioethanol by which the production can be carried out even in a high latitude region having little irradiation of sunlight in a low-temperature environment by utilizing a surplus quantity of heat in the industrial production and further utilizing an organic waste and discharged carbon dioxide by dragging the faint sunlight thereinto by using an optical fiber. <P>SOLUTION: The method for continuously producing bioethanol includes using starch particles continuously obtained by continuously culturing alga without collecting the algae cells (alga body) themselves by allowing starch particles to be released to the exterior of the cells, and subjecting the starch particles to saccharification and fermentation steps. Bioethanol can be produced even in the whole region of from the tropical region to the high latitude region in the world, and bioethanol having a cost meeting a price as an alternate fuel can be produced by the reduction of the production cost by carrying out the successive starch production. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for producing starch using algae that release starch extracellularly. Furthermore, this invention relates to the manufacturing method of ethanol using the algae which discharge | releases starch outside a cell.

  From the end of the last century, fossil fuel depletion and the increase in carbon dioxide emissions caused by fossil fuel combustion have been screamed. Therefore, it is desired to produce so-called bioethanol as a fuel by fermenting a polysaccharide such as starch obtained by fixing carbon dioxide (hereinafter referred to as carbon dioxide) to a plant. Has been done. However, the method of using cereals used as food and feed as raw materials will inevitably become a major problem in the future, as food prices rise and the number of people suffering from hunger increases. Therefore, carbon dioxide can be fixed by photosynthesis in the same way as general plants, and algae with high biomass productivity that can efficiently produce starch is obtained, and ethanol is fermented and produced from biomass obtained using this. Has been expected to.

As for general microorganisms, foods and pharmaceuticals obtained by fermentation engineering, and their additives are widely used in general, and a wide variety of enzymes, etc. obtained from microorganisms are industrially used. Used in the field. However, the use of algae means that in Japan, large algae such as kombu, wakame, and seaweed are commonly eaten, and microalgae such as chlorella are used as health foods. The total amount is very small. In addition, substances obtained from these algae are in a state of being hardly used except that polysaccharides such as agar, alginic acid and carrageenan are used as thickeners and gels. . Research on algae has just started, centering on research on photosynthesis, and algae is rarely used industrially. This originates from the fact that the growth rate of algae has been extremely slow compared to conventional microorganisms.

Algae are widely distributed on the earth, from fresh water to seawater, and to special environments such as hot spring sources, have evolved into a wide variety of forms and biological characteristics, and are well adapted to the global environment. Many algae can grow in the same manner as normal microorganisms under heterotrophic conditions in the dark, in addition to growing under autotrophic conditions by light irradiation. This is different from higher plants that grow only under photosynthetic autotrophic (hereinafter referred to as photoautotrophic) conditions and are considered to be the most prosperous and most evolved on the ground. Utilizing the characteristics of this algae, carbon dioxide and organic waste that are discharged industrially are used as nutrient sources, and sunlight irradiation is performed to make them photoautotrophic. It has been desired to efficiently culture algae and efficiently produce useful biomass such as starch.

Like plants, carbon dioxide fixation by photosynthesis, or using organic matter as a carbon source, efficiently producing starch, accumulating a large amount of starch granules in the cell, and releasing a part of it outside the cell, An excellent algae strain, a single-cell fine green algae, Chlorella vulgaris Al-1y-3 (hereinafter also referred to as Chlorella vulgaris Al-1y-3) is known (Patent Document 1). Moreover, a method for producing ethanol using starch obtained from algae in this way as a raw material is known (Patent Document 2).

In recent years, research and development of bioethanol has been actively performed, and methods using seaweed algae (Patent Document 3) and (Patent Document 4) are also known, and these are industrial wastes. A method using combustion ash as an inorganic nutrient source has also been reported.
JP-A-52-82793 JP 50-148587 A JP 2000-316593 A JP2003-310288

  In the conventional technology, algal cells are killed at the stage of fermenting the obtained starch with ethanol, so the production process is not continuous, which leads to high costs and has not actually sold bioethanol. .

  As a result of diligent efforts to eliminate the drawbacks of the prior art, the present inventors have found a method for continuously obtaining starch produced by algae by culturing continuously without killing algal cells. Completed the invention.

That is, the present invention relates to a method for producing starch, which comprises culturing without killing algal cells and collecting algal cells. Preferably, the present invention is a method for producing starch comprising:
(A) culturing an algae that releases starch outside the cell;
(B) It is related with the method including leaving the culture | cultivation still and isolate | separating the released starch and algae, and (c) collect | recovering the isolate | separated starch.
More preferably, in addition to the above (a)-(c),
(D) It relates to a method for producing starch comprising the step of further culturing algae left unrecovered in step (c), and (e) repeating the steps (b) to (d) above.
More preferably, the step (e) is a step of continuously repeating the steps (b) to (d).

  The present invention also relates to a method for producing ethanol comprising culturing without killing algal cells and collecting algal cells. Preferably, the present invention is a method for producing ethanol, comprising the steps of saccharifying the produced starch, and subjecting the saccharified starch to alcohol fermentation after producing the starch by the method for producing starch according to the present invention. About. More preferably, the ethanol production method includes a step of irradiating the starch with microwaves in a closed pipeline before the step of saccharifying starch or during the step of saccharifying starch.

Further, in the method for producing starch and ethanol, the step (a) of culturing the algae is performed by photoautotrophic by carbon dioxide and light irradiation, or by heterotrophic by adding organic matter, carbon dioxide and light. Including culturing by photoautotrophic irradiation by irradiation in parallel, as carbon source (nutrient source), use acetic acid, organic acid waste liquid, industrially discharged carbon dioxide (carbon dioxide) or carbonate Can do.
In addition, the step (b) of separating the released starch and algae includes separating the starch and the algae by decantation or water flow.

  The algae used in the method for producing starch and the method for producing ethanol according to the present invention are not limited as long as they are algae that release starch to the outside of the cells, preferably chlorella, more preferably, Chlorella vulgaris Al- 1y strain.

  Since the method for producing starch and ethanol according to the present invention can be continuously cultured without killing algal cells, starch and ethanol can be produced from algal cells at a lower cost than conventional methods.

As used in the present invention, `` algae that release starch outside the cell '' is an algae that releases, excretes, or secretes starch outside the cell if it is a unicellular organism, and outside the alga if it is not a unicellular organism, Although not particularly limited, Chlorella vulgaris Al-1y strain (hereinafter also referred to as Chlorella vulgaris Al-1y strain) (Accession Number: FERM BP-10915) is preferable.
The culture conditions are not particularly limited, and the composition of the medium can be appropriately set depending on individual algae. For example, organic substances (for example, organic acids such as acetic acid, formic acid and citric acid, sugars such as glucose and maltose, and / or lipids) as a carbon source, and organic or inorganic substances (for example, urea, ammonium sulfate, ammonium acetate, ammonium nitrate) as a nitrogen source , Potassium nitrate, polypeptone, etc.) and a medium containing inorganic salts can be used, and carbon dioxide (carbon dioxide), carbonates and the like can also be used as a carbon source.
In addition, conditions such as algae, in particular, pH of the medium for culturing chlorella, culture temperature and the like can be appropriately set by those skilled in the art, but are preferably pH 5-9, culture temperature 25-42 ° C., more preferably, pH 7-8, culture temperature 30-37 ° C.
Moreover, as culture conditions (including medium composition, culture temperature, etc.), conditions under which cell division is actively performed are preferable.

  Using the above medium, algae can be cultivated aerobically in the dark (heterotrophic culture) or autotrophic by photosynthesis. Furthermore, algae can be cultured in parallel with heterotrophic culture (culture with heterotrophic nutrition) and autotrophic culture (culture with photoautotrophic nutrition). For example, algae can be cultured heterotrophically using a medium supplemented with organic substances (organic acids, sugars and / or lipids) as a carbon source, and carbon dioxide is used as a carbon source for light irradiation. Can also be cultured in an autotrophic manner. In some cases, the culture may be performed by coexisting the culture by heterotrophic with the addition of the organic substance and the culture by photoautotrophic with carbon dioxide and light irradiation. In addition to sunlight, artificial light such as a light bulb or an optical fiber can be used as light necessary for photosynthesis.

  From the viewpoint of contribution to the environment and the production of inexpensive starch and ethanol, the organic matter as a nutrient source can be a commercially available product, but a waste liquid or the like can also be used. In the starch production process using algae, it is possible to produce ethanol at a very low cost by using, as a carbon source, organic acid waste liquid that has been industrially discharged in large quantities. The type of organic acid waste liquid to be added as a carbon source and the addition method are not limited, but acetic acid is widely used industrially and has a large discharge amount. As an example of an organic acid waste liquid that can be easily used. Examples include acetic acid waste liquid. In addition, waste liquids of glycerin and alcohols can be used. These waste liquids may be used alone or in combination. Further, carbon dioxide (hereinafter also referred to as carbon dioxide) or carbonate discharged industrially can be used as the carbon source. As so-called exhaust gas, the amount of carbon dioxide discharged has been increasing year by year, and it has been screamed to reduce carbon dioxide emissions, but it is extremely difficult to implement, and it is impossible to reduce carbon dioxide emissions. It is said that there is. Conventionally, there is no method for reducing the low-concentration carbon dioxide gas released into the atmosphere and diffused under natural conditions other than by plants. The present invention obtains ethanol by reusing industrially discharged high-concentration carbon dioxide gas without releasing it into the atmosphere, and can significantly reduce carbon dioxide emission. As a method, industrially discharged carbon dioxide gas may be directly ventilated into the algae culture tank. However, when a decrease in pH is observed due to carbon dioxide gas ventilation, an alkaline agent such as sodium hydroxide, or industrial It is also possible to neutralize by using ash wastes that have been discharged. As a nitrogen source to be added to the culture medium, pig manure discarded from livestock farmers, other livestock, poultry waste, slaughterhouse and slaughterhouse waste can be used alone or in combination. good.

  The separation of the released starch and algae in the present invention is not particularly limited, but the physical properties such as the particle size and / or shape of starch and algae cells by centrifugation, sieving, decantation or water flow, etc. It can be done by separation means based on. The present inventors have cultured algae that release starch outside the cell, especially chlorella that releases starch outside the cell, and if left standing for a sufficiently long period of time, on the algae sedimentation layer that has settled in the incubator, It was found that starch particles released from algae accumulate. The standing time varies depending on the size and shape of the incubator and the algae to be cultured, but is 1 to 24 hours, preferably 3 hours or more.

  After standing, the starch particles naturally settled and deposited on the layer of algae are white and can be clearly distinguished from algae such as dark green. Starch can be easily separated from algae by decantation or water flow and collected. can do. Such easy separation of starch and algae is not necessary especially in the industrial culture of chlorella with a large amount of culture solution, and a device such as a centrifuge or sieve that can handle a large amount of medium is not required. Contributes to the reduction of manufacturing costs. In addition, after culturing, the cultured algae remain in the incubator by collecting and recovering the starch that naturally settled by standing and deposited on the algae by decantation, etc., and the culture medium is added to this to recover the algae It is possible to easily re-culture without doing.

  In the present invention, the means for recovering the separated starch is not particularly limited. For example, the starch may be recovered by washing with a solvent such as water or acetone, and centrifuging at 2000 rpm with a centrifuge, for example. it can.

The step of further culturing the algae left uncollected after collecting the starch is achieved, for example, by re-adding the medium to the algae left in the incubator and re-culturing under the initial culture conditions. obtain. The medium and culture conditions can be appropriately set by those skilled in the art as described above. Thereafter, the culture is allowed to stand still, and the process of separating released starch and algae, the process of collecting the separated starch, and the process of further culturing the remaining algae without being collected are repeated continuously. In addition, starch and ethanol can be produced from algae.
The present inventors have found that the amount of starch obtained by culturing algae that releases starch outside the cell, especially chlorella that releases starch outside the cell, does not change greatly. In addition, even when the amount of chlorella at the beginning of the culture is higher than at the time of the initial culture at the time of re-culture, the same culture medium is used in the same incubator as the previous culture, so that the same growth as the previous culture is performed. And found that re-cultivation is possible up to at least 7 times. Considering that starch is released extracellularly during cell division, it can be continuously grown at least 8 times in the same incubator using the same medium volume without recovering chlorella, It was unexpected that almost the same amount of starch could be stably recovered with the number of cultures. Moreover, in the re-culture, the culture time until the end of the logarithmic growth phase was shortened compared to the initial culture. Such shortening of the culture time is one of advantageous effects because it lowers the production cost of starch and ethanol.

In the present invention, ethanol can be produced by saccharifying the algal starch recovered as described above and subjecting it to alcohol fermentation. The method for saccharification and alcohol fermentation is not limited, but as an example, the saccharification method includes acid saccharification method, enzymatic saccharification method, etc. Other microorganisms such as bacteria having alcohol-producing ability may be used.
For example, for saccharification of starch, starch saccharifying enzymes, such as Magnax JW-101 (including Nakto Kasei Kogyo Co., Ltd., Magax JW-101, RAKUTO KASEI INDUSTRIAL Co., Ltd .: including α-amylase and glucoamylase) The saccharification treatment may be performed biochemically by using a method such as enzymatic saccharification of starch and alcohol fermentation simultaneously (parallel fermentation). In addition, saccharification treatment and alcohol fermentation may be simultaneously performed by a method of performing enzymatic saccharification of starch by microorganisms such as mold mold and alcohol fermentation simultaneously, so-called parallel re-fermentation.

  Microwave irradiation may be performed in a closed pipeline as a pretreatment for fermentation or at the time of saccharification of starch. In general, microwaves are electromagnetic waves in the range of 100 μm to 1 m and frequencies of 300 MHz to 3 terahertz. In Japan, frequencies of 2.45 GHz are used for heating devices such as microwave ovens, and in the United States, 9.15 MHz is also used in addition to this. . In the present invention, although the wavelength (frequency) of the microwave is not limited, it is inexpensive to use a frequency of 2.45 gigahertz, which is widely used in general. In order to produce ethanol from starch, a conventional method requires a saccharification step of starch using an acid or an enzyme. This saccharification step requires heat treatment, and in the conventional technology, direct heating by a heat source has been performed. However, when the capacity becomes large, a long time and a large amount of heat energy are required for temperature increase and decrease. It was. The present invention is not limited regarding the method for saccharification of starch, but it is possible to instantaneously raise and lower the temperature of starch granules obtained from an algal culture tank in a closed pipeline. By performing irradiation, heat treatment can be performed very effectively.

  After alcohol fermentation, the ethanol obtained can be distilled and dehydrated to produce alcohol with increased alcohol concentration. The means for distillation and dehydration are not particularly limited, and can be appropriately selected from means generally known to those skilled in the art.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. The technical scope of the present invention is not limited by the following embodiments, and can be implemented with various modifications without changing the gist thereof.

According to the present invention, the cultured algal cells (algae) are continuously cultured without collecting and killing the algae itself, and continuously obtaining the starch granules released by the algal cells. As shown in FIG. 1, there are five methods for producing bioethanol: algae culture step (S1), starch separation step (S2), starch saccharification step (S3), alcohol fermentation step (S4), and distillation step (S5). Steps may be included. The algae culture step (S1) can include three types of culture methods, ie, photoautotrophic (S11), heterotrophic (S12), and mixed nutrition (S13). Photoautotrophic (S11) is a culture method based on photosynthesis that uses inorganic salts and carbon dioxide as a carbon source (nutrient source), as in the case of general higher plants. The yield per area is low. Moreover, when sunlight is used as a light source, no nighttime proliferation can be expected. Usually, the cultivation of algae under photoautotrophic conditions by photosynthesis uses nitric acid or ammonia as a nitrogen source, and phosphoric acid and magnesium are added about 1/5 to 1/20 of the nitrogen source, respectively. In addition, a medium prepared by adding a small amount of various metals including a carbon dioxide gas is aerated. Heterotrophic (S12) is a culture in which an organic substance is added to a medium as a carbon source, and can be cultured at a high density in a completely dark state and has a high yield per unit area. Mixed nutrition (S13) is a method (mixotrophic) in which both the steps of S11 and S12 are combined, that is, culture by heterotropy by adding organic substances and photoautotropy by carbon dioxide and light irradiation are performed in parallel.
The culture method based on each of these nutrient conditions is appropriately selected according to the location conditions of the culture apparatus, depending on the natural environment such as temperature (air temperature) and sunshine duration, the type of waste used, and the like.

  The starch separation step (S2) is a step of separating and recovering starch released to the outside by the algae sufficiently grown in the algae culture step (S1). Examples of the separation method include a separation method by natural sedimentation (S21), a method by centrifugation (S22), and a separation method by water stream (S23).

  The starch saccharification step (S3), the alcohol fermentation step (S4), and the heat treatment step (S5) can be performed as separate and independent processes, but can be performed in parallel and are usually performed in parallel. Implemented as a process. S3 is carried out by microorganism saccharification method (S31), enzyme saccharification method (S32), acid saccharification method (S33), etc., and alcohol fermentation step (S4) is a microorganism such as alcohol yeast or alcohol-fermenting bacteria. Can be implemented. When step S33 is performed, a neutralization step (S34) is required. The acid and alkali used in these steps are not particularly limited. S5 can be performed when starch is pregelatinized prior to S31 or S32 and when hydrolysis of S33 with acid is performed. This heat treatment may be performed by microwave irradiation in a closed pipeline. By using a frequency of 2.45 gigahertz (usually 9.15 megahertz can also be used in the United States), which is widely used, costs can be reduced in industrial production.

  Ethanol obtained by the above production method has an alcohol concentration of 5 to 20%, and the alcohol concentration can be increased by the distillation step (S6). Distillation methods include atmospheric distillation by simple heating (S61), vacuum distillation performed at room temperature or under reduced pressure (S62), and vibration distillation performed by accelerating vaporization by oscillating with ultrasonic waves, etc. (S53) and the like are included, and these methods may be performed alone or in combination (S64).

  Bioethanol used as a fuel for an internal combustion engine such as an automobile must have a low water content as much as possible in order to avoid adverse effects on the internal combustion engine. Therefore, in some cases, a dehydration step (S7) is added. The dehydration method uses anhydrous salts such as anhydrous sodium sulfate, metal oxides such as quicklime, or other chemicals (S71), water absorption using porous materials such as zeolite as they are or after high-temperature treatment (firing). Method (S72), a method using a water-absorbing material such as silica gel (S73), and the like, and a method combining these methods (S74) can also be employed. In the laboratory, a method using molecular sieve is simple.

(Example 1)
Algae such as Chlorella vulgaris Al-1y was cultured, and the relationship between algal cell weight and the amount of starch obtained was examined.

In 1 L of liquid medium used for algae culture, 2 g of sodium nitrate (NaNO ₃ ), 0.2 g of magnesium sulfate (MgSO ₄ · 7H ₂ O), 50 mg of calcium chloride (CaCl ₂ · 2H ₂ O), 25 mg ferrous sulfate (FeSO ₄ .7H ₂ O), dipotassium hydrogen phosphate 0.8g (K ₂ HPO _4), dihydrogen phosphate monopotassium 0.25g (KH2PO4), boric acid 3mg (H3BO3), 2 mg manganese chloride (MnCl2 · 4H ₂ O), 500 µg cobalt nitrate (Co (NO ₃ ) ₂ · 6H ₂ O), 20 µg zinc sulfate (ZnSO ₄ · 7H ₂ O), 8 µg copper sulfate (CuSO ₄ · 5H ₂ O), 2 μg of sodium molybdate (Na ₂ MoO ₄ · 2H ₂ O). To this liquid medium, 15 g each of agar and glucose were added, dissolved by heating, and solidified slant agar medium was used as a storage medium for algae. In addition, liquid culture for algae growth was performed in the dark under heterotrophic conditions by adding 20 g of ammonium acetate, 8 g of sodium acetate, and 7 g of potassium acetate as carbon sources to the above medium composition. During the cultivation, the pH fluctuated to the alkali side with the growth of algae. For the purpose of correcting this, acetic acid was added and the pH was always adjusted and maintained within the range of 6.5 to 7.5. The medium dispensed in all containers was steam sterilized at 121 ° C. for 15 minutes.

Chlorella vulgaris Al-1y strain on the storage medium was transplanted to 10 ml of algae growth / liquid medium in a 100 ml baffled Erlenmeyer flask with a vented silicon stopper and shaken on a rotary shaker set at 35 ° C. for 72 hours (Number of shaking: 110 times / minute). The whole amount was transplanted to 90 ml of the same medium in the same 500 ml flask (total liquid volume: 100 ml) and cultured under the same conditions for 72 hours. This was transplanted to 900 ml of the same medium in a small fermentor (jar fermenter) with a volume of 1.5 L (total liquid volume: 1 L), aerated with the same volume of air as the medium volume per minute, and agitated at 100 rpm. In the same manner, the cells were cultured for 96 hours. Algae cell growth is confirmed by turbidity measurement by absorbance measurement at 540 nm every 8 hours and measurement using a Birkel-Turck hemocytometer, and transplantation is performed immediately before the end of the logarithmic growth phase. It was. After completion of the culture, algal cells and released starch granules were collected by centrifuging 2,000 g for 10 minutes, dried at 105 ° C., and weighed. The amount of starch contained in the algal cells is calculated by subtracting the quantitative amount of reducing sugars by the somoegi-Nelson method from the quantitative amount of total sugars by the anthrone-sulfuric acid method. The multiplied value was defined as a quantitative value. In addition, it was confirmed by the iodine starch reaction that what was recovered was starch.

As shown in FIG. 2 and FIG. 3, the growth of chlorella reached a maximum value after 56 hours of culture, and the dry weight of algal cells in 1 L of the culture solution 96 hours after the start of the culture is 7.5 g. The amount of starch was 5.6 g. About 75% of the algal cell weight is present as starch, which is due to the accumulation of starch granules released from the cells of the Chlorella vulgaris Al-1y strain used in this example during culture. . In experiments conducted using other types of green algae strains, a dry weight of algae cells of 8.5 g was obtained for Chlamydomonas sp. Al-5 and 8.8 g for Scenedesmus basilensis C-66. The starch content was low, about 30%. From the results of this Example, the excellent starch production capacity of the Chlorella vulgaris Al-1y strain was confirmed. Therefore, in Example 2 shown below, the algae cells of the Chlorella vulgaris Al-1y strain were continuously maintained to produce starch.

(Example 2)
As in Example 1, Chlorella vulgaris Al-1y strain was cultured in a 200 L culture tank (fermenter) using 100 L medium, and after the logarithmic growth phase was completed and a stationary phase was reached, aeration was performed. Then, the stirring was stopped, and the algal cells and starch granules were separated by decantation by natural sedimentation for 16 hours. The obtained starch granules were dehydrated by centrifugation, added with acetone, dried at room temperature under reduced pressure, and 97 g of dried extracellular released starch granules were obtained. Moreover, since the culture solution containing the separated algal cells was 1/10 of the liquid volume (about 10 L) before the separation operation, 90 L of the culture medium used in Example 1 was added thereto, As the culture volume (100 L), aeration and agitation of the culture tank were resumed, and after culturing for 32 hours, starch granules were collected in the same manner as described above. And the cell which isolate | separated and collect | recovered starch was further culture | cultivated. Similarly, the culture was continuously repeated to collect starch granules. Table 1 shows the number of times of cultivation and the obtained starch granule weight.

Thus, although there was some fluctuation, since the starch granules could be stably produced continuously, ethanol was produced on trial using the obtained starch as a raw material. Suspend 500 g of starch granules obtained by the above method in 2.5 L of water containing 50 ml of concentrated sulfuric acid, and pass a 10 mm diameter Teflon (registered trademark) tube at a flow rate of 50 mm / sec. Irradiated with an output of 1.1kW, the product temperature was maintained at 90 ° C or higher for 30 minutes, and starch was saccharified. This saccharification by irradiation is confirmed by measuring the amount of reducing sugars by the Somogy-Nelson method, adjusted to pH 6 by adding lime milk (calcium hydroxide: Ca (OH) ₂ suspension), and then magnesium sulfate ( MgSO ₄ · 7H ₂ O) 0.02%, dipotassium hydrogen phosphate (K ₂ HPO ₄ ) 0.2%, and urea ((H ₂ N ₂ ) CO) 0.1% were added to make a medium for fermentation. . 500 ml of yeast (Japan Brewing Association / Association No. 7, Saccharomyces cerevisae Kyokai No. 7, Brewing Society of Japan) previously cultivated using rice bran medium is added to the fermentation medium with a sugar concentration of about 20%. Then, static culture was carried out at 30 ° C. until the reducing sugar disappeared (7 days).

  The ethanol-containing fermented solution obtained by alcohol fermentation with yeast was distilled twice at 50 ° C. under reduced pressure using a rotary evaporator. When the ethanol concentration of this distillate was measured with a float-type hydrometer, it was 72% and the amount of liquid obtained was 306 ml. To this ethanolic distillate, 150g of molecular sieves previously dried in dry heat was added and allowed to stand for 24 hours, and then distilled again under the same conditions.To the obtained distillate, 20g of molecular sieves previously dried in dry heat was added and allowed to stand for 3 days. 194 ml of 99% or more ethanol was obtained.

(Example 3)
The following industrial wastes were used as raw materials for bioethanol production. As a carbon source, acetic acid waste liquid discharged from a chemical factory with an acetate ion concentration of about 20% (pH 2.5-4.0) and as a nitrogen source, pig manure discarded from livestock farmers is recovered. Then, a liquid obtained by removing the solid matter from the residue obtained by recovering the carbon source as methane gas by subjecting this to methane fermentation and containing about 0.5% as ammonia nitrogen was used. Also, assuming carbon dioxide gas burned and discharged from the refining process of oil and natural gas, the carbon dioxide concentration was adjusted to 10 times that of the atmosphere and ventilated.

The above waste liquid was mixed and adjusted to contain about 2% acetate ion and about 0.1% ammonium ion. Magnesium sulfate (MgSO ₄ · 7H ₂ O) 0.01%, dipotassium hydrogen phosphate (K ₂ HPO ₄ ) Add 0.008%, monosodium dihydrogen phosphate (NaH ₂ PO ₄ ) 0.002%, and vent the air containing the above carbon dioxide gas per minute at the same volume as the medium liquid volume, and the pH is always 6 ~ It adjusted using the acetic acid waste liquid so that it might be in the range of 8. A culture solution in which the Chlorella vulgaris Al-1y strain was shaken (precultured) for 72 hours in a flat glass flask (culture solution volume: 400 ml) filled with this waste liquid medium and having an inner height of 220 mm, a width of 100 mm, and a thickness of 20 mm. Was transplanted in 1/10 volume of the medium volume. By culturing 30 such culture media (total culture volume: 12L) for 6 days in an artificial meteor with illuminance of 4,000Lux, adjusted to 35 ° C under fluorescent light irradiation, Ascertained, the culture was terminated, and algal cells and released starch granules were collected.

  According to the same analysis method as in Example 1, the dry weight of algal cells contained in 12 L of the culture broth was 87 g, the amount of starch contained in the cells was 36 g, and the starch granules released outside the cells were measured as 19 g. . 10 g of the obtained starch granules were saccharified in the same manner as in Example 2, and production of alcohol was confirmed using an apparatus for alcohol fermentation test. In alcoholic fermentation, equimolar (same number of molecules) of carbon dioxide as the produced ethanol is discharged, so this test device traps water vapor generated in the incubator with concentrated sulfuric acid and releases carbon dioxide. The weight reduction is weighed to investigate the amount of ethanol produced, and is used for a small amount of testing. As a result, a weight reduction of 3.9 g was observed, indicating that about half of the starch weight was obtained.

1 is a flow diagram illustrating a method for producing bioethanol according to a preferred embodiment of the present invention. The growth curve of Chlorella vulgaris Al-1y strain | stump | stock in a 1.5L small fermenter (jar fermenter) measured by the turbidity method (540 nm light absorbency measurement) in Example 1. FIG. The growth curve of the Chlorella vulgaris Al-1y strain | stump | stock in the 1.5L small-sized fermenter (jar fermenter) measured using the Birkel-Churk type hemocytometer in Experimental example 1. FIG.

Explanation of symbols

S1 Primary process, algae culture process
S11 Light autotrophic
S12 heterotrophic
S13 Culture by mixed nutrition
S2 Secondary process, starch separation process
S21 Separation method by natural sedimentation
S22 Centrifugal method
S23 Water separation method
S3 Tertiary process, starch saccharification process
S31 Saccharification by microorganisms
S32 Enzymatic saccharification method
S33 Saccharification method with acid, etc.
S34 Neutralization process
S4 Quaternary process, alcohol fermentation process
S5 Fifth process, heat treatment process
S6 Sixth process, distillation process
S61 atmospheric distillation
S62 vacuum distillation
S63 Vibration distillation method
S64 Distillation method combining these methods
S7 Dehydration process
S71 Dehydration method by drug absorption
S72 Dehydration by porous water absorption
S73 Dehydration method using water-absorbing material
S74 Dehydration method combining these methods

Claims (11)

A method for producing starch, comprising:
(A) culturing an algae that releases starch outside the cell;
(B) A method comprising standing the culture and separating the released starch and algae, and (c) collecting the separated starch. further,
The method according to claim 1, comprising (d) further culturing algae left unrecovered by step (c), and (e) repeating steps (b) to (d). After producing starch by the method according to claim 1 or claim 2,
(F) The method of manufacturing ethanol including the process of saccharifying the starch collect | recovered by Claim 1 or Claim 2, and the process of (g) alcohol-fermenting the saccharified substance of starch. further,
(H) a step of irradiating the starch with microwaves in a closed pipeline before the step of saccharifying starch or during the step of saccharifying starch;
The method of manufacturing ethanol of Claim 3 containing this. The step (a) of culturing the algae includes culturing by photoautotropy by carbon dioxide and light irradiation,
The method to manufacture the starch or ethanol in any one of Claim 1 to 4. The step (a) of culturing algae includes performing culture by heterotrophic by adding organic matter and culture by photoautotrophic by carbon dioxide and light irradiation,
The method to manufacture the starch or ethanol in any one of Claim 1 to 4. The step (a) of culturing the algae is performed using an organic acid waste liquid as a carbon source.
A method for producing the starch or ethanol according to any one of claims 1 to 6. The step (a) for culturing algae is performed using carbon dioxide (carbon dioxide) or carbonate that is industrially discharged as a carbon source.
A method for producing the starch or ethanol according to any one of claims 1 to 6.   The method for producing starch or ethanol according to any one of claims 1 to 8, wherein the step (b) of separating released starch and algae includes separating starch and algae by decantation or water flow.   The method for producing starch or ethanol according to any one of claims 1 to 9, wherein the algae is chlorella.   The method for producing starch or ethanol according to claim 10, wherein the chlorella is Chlorella vulgaris Al-1y strain.

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