JP2013039085A - Method for producing ethanol - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing ethanol in which a supply cost of essential nutrients and required trace elements of fermentation microorganisms is low by using vegetable wastes as a raw material of biomass ethanol.SOLUTION: A saccharified product of a vegetable polysaccharide and a microalgae collapsed product in which protoplasm is released to the outside of a cell are prepared to produce ethanol by ethanol-fermenting the saccharified product using a microorganism in the presence of the microalgae collapsed product. As a supply resource of the essential nutrients and required trace elements of the microorganism, the protoplasm released from the microalgae collapsed product is used.

Description

  The present invention relates to a method for producing ethanol, and in particular, relates to a method for producing ethanol by producing biomass ethanol by fermentation using microorganisms using plant waste such as wood or wheat straw.

  In recent years, biomass ethanol, which produces ethanol by fermentation of microorganisms using plant matter, has attracted attention as a solution to the depletion of petroleum resources, and production has actually started. However, the raw material of biomass ethanol currently produced is cereals containing starch and saccharides such as corn, and this is a useful material for food. There is a problem above.

  For this reason, the production of biomass ethanol using plant waste has been studied, and woody waste materials such as building materials, plant matter such as wheat straw, rice straw, and corn stover (stems and leaves excluding corn) Development of technology for ethanol fermentation of cellulose using waste as a raw material is underway.

  In order to produce biomass ethanol, monosaccharides such as glucose are required for the yeast that performs ethanol fermentation to be active and proliferate, so enzymes are added to break down cellulose in plants and supply monosaccharides. There are other nutrient sources necessary to maintain normal yeast activity, and yeast extract and polypeptone are used in the production of ordinary fermented foods.

  However, when yeast extract or polypeptone is used as a nutrient source for yeast, the production cost increases, so that it is not suitable for commercial production of biomass ethanol. For these reasons, the use of various plant wastes as substitutes has been studied. For example, the following Patent Document 1 proposes adding okara, tea bowl, coffee bowl and the like.

Japanese Patent No. 4401735

  The food waste such as okara proposed in Patent Document 1 contains a lot of water and is easily rotted. Therefore, in order to use these, the storage environment such as refrigeration or the like is used to prevent rot. And the transportation cost increases due to the weight due to the large amount of water. In addition, food processing factories that produce the above food waste are often located close to consumption areas such as metropolitan areas, and long-distance transportation is not preferable to prevent corruption. Manufacturing facilities need to be installed near the city. However, it is preferable to install biomass ethanol production facilities in the vicinity of forests or large-scale farms where vegetable waste, which is the raw material for production, is produced, and costs related to transportation and storage for using food waste, etc. It is difficult to solve the problem. Furthermore, since the amount of food waste varies depending on the degree of preference for food, depending on the season and national character, stable raw materials are difficult depending on the season and may not be available overseas.

  The object of the present invention is to solve the above-mentioned problems and to produce biomass ethanol stably from cellulose as a vegetable waste with low cost required for supplying nutrients and the like necessary for maintaining the activity of fermentation microorganisms normally. It is to provide a method for producing ethanol.

  In addition, other problems of the present invention are not limited to seasons and national patterns, and supply nutrients and the like necessary for maintaining normal activity of fermenting microorganisms through a supply route suitable for the location conditions of the biomass ethanol production facility. It is to provide a method for producing ethanol.

  In order to solve the above-mentioned problems, the present inventors have conducted extensive research and found that nutrition and the like necessary for maintaining the activity of fermentation microorganisms normally can be supplied using algae. The present invention has been completed.

  According to one aspect of the present invention, a method for producing ethanol comprises preparing a saccharified product of a plant polysaccharide and a microalgae disintegrated material whose cytoplasm is released to the outside of the cell, and in the presence of the microalgae disintegrating material. The gist is that, by subjecting the saccharified product to ethanol fermentation using a microorganism, the protoplasm released from the microalgae collapse can be used as a source of essential nutrients or required trace elements of the microorganism.

  ADVANTAGE OF THE INVENTION According to this invention, the manufacturing method of ethanol which can supply the nutrient source required in order to maintain the activity of fermentation microorganisms normally without being restrict | limited to a season, a national pattern, and location conditions is provided, and the manufacturing facility of biomass ethanol Since it can be carried out easily according to the location conditions, it is economically advantageous. In addition, utilization of plant waste as biomass is promoted, which is useful for solving the waste disposal problem.

  In the production of biomass ethanol using plant waste, ethanol fermentation is carried out on a saccharified product obtained by hydrolysis of plant cellulose. In the process of ethanol fermentation, not only simple sugars such as glucose but also essential nutrients such as phosphorus, nitrogen and vitamins, and required trace elements such as Co, Ni, and Zn are necessary for yeast to be active and proliferate. Yeast uses these to biosynthesize proteins and nucleic acids. In addition, depending on the type of yeast, vitamins and some amino acids cannot be synthesized by themselves, so it is necessary to add them from the outside. Yeast used to produce biomass ethanol has a low or lack of ability to synthesize vitamins or amino acids, etc., and ethanol in a medium containing only glucose and inorganic salts (phosphorus, nitrogen, calcium, magnesium, etc.) In order to significantly reduce the production rate and maintain the yeast activity normally, it is necessary to add an expensive yeast extract, polypeptone, or the like to supply organic components. In particular, when plant waste cellulose is used as biomass, it is important to supply the above-mentioned organic components, vitamins and inorganic salts.

  In the present invention, a protoplasm (particularly cytoplasm) that is a cell content of microalgae is used as a source of the above-mentioned organic components and inorganic salts that are essential nutrients and required trace elements of fermentation microorganisms. Various organic components and minerals contained in the traits are supplied to the fermentation system in a state where microorganisms can easily be taken up. That is, the method for producing ethanol in the present invention provides a saccharide saccharification product prepared using plant waste, and a microalgae disintegration product whose protoplasm is easily released outside the cell, The saccharified product is ethanol-fermented by microorganisms in the presence of microalgae collapse. Below, the manufacturing method of ethanol of this invention is demonstrated in detail.

  In algae, some seaweed is used as food, but the use of microalgae is relatively limited. For example, microalgae such as Chlorella, Nannochloropsis, Donariella, Botriococcus, Spirulina, Schizochytrium, Aurantiochytrium, Keitoseros calcitrans, Keitoseros gracilis, Pavlova luteri, Isocrisis galvana, etc. It is only used as a raw material for food, aquatic organisms, etc., but it is promising as a source of essential nutrients and required trace elements for fermenting microorganisms because the protoplasm contains abundant ingredients useful for the growth of organisms. Therefore, it is useful as biomass and does not cause an increase in the price of food raw materials and the like when promoting the use of microalgae. In addition, there are two types of microalgae: one that grows in seawater and the other that grows in freshwater. Select the appropriate type according to the location environment of the biomass ethanol production facility and obtain microalgae using seawater or freshwater. Therefore, it can be used at any location in the coastal area, plains, and mountains, and does not limit the location of biomass ethanol production facilities.

  In the present invention, the present invention is not limited to the microalgae exemplified above, but can be appropriately selected from microalgae containing components useful for the growth of microorganisms and can be used after being grown according to a known culture method. . For example, in the case of Chlorella, water containing organic matter is used as a culture solution and cultured aseptically while aerated under dark conditions. If necessary, inorganic salts such as ammonium chloride and potassium phosphate are added, so that it grows properly. (Reference: For example, Japanese Patent Laid-Open No. 5-284962). Chlorella grown in fresh water grows on organic matter such as glucose and acetic acid, regardless of photosynthesis. In such microalgae culture, the distillation residue after separation and recovery of ethanol from the fermentation broth is used as the culture solution. When used, the glucose remaining in the residual liquid can be effectively used, so that the efficiency is high, and it is advantageous that it is in a sterilized state by heating during distillation. Marine microalgae such as Nannochloropsis are used as a culture solution by filtering seawater with a filter as necessary and killing other microorganisms by ultraviolet irradiation. The cultured microalgae may be appropriately concentrated and used using a filter having a pore size smaller than the size of algal cells (generally about 4 μm or more). Alternatively, microalgae that naturally propagated in lakes, rivers, harbors, dams, water areas in the vicinity of power generation facilities, etc. may be collected and used. It can be used after being subjected to sterilization such as ultraviolet irradiation.

  In order for fermenting microorganisms to incorporate the components in the protoplasts of microalgae obtained by culture or collection, the protoplasm must be released to the outside of the cell, so the cells of microalgae are disrupted, that is, the physical or chemical nature of the cell membrane or cell wall. After the cell is unwrapped by performing general tearing, decomposition, or lysis and processed into a collapsed state in which the protoplasm is likely to flow out of the cell, this microalgae disintegration product is put into the fermentation system. Thereby, yeast etc. which are fermentation microorganisms can carry out ethanol fermentation of the saccharide | sugar saccharified plant polysaccharide, taking in the essential nutrients etc. which are contained in the protoplast of a micro algae, and utilizing them for proliferation and growth. The microalgae disintegrates need only be in a state in which the protoplasm can be released, that is, in a state in which the protoplasm can be easily released out of the cell, and it is necessary to prepare the protoplasm in a state in which the protoplasm is completely released outside the cell. Absent. Further, it is not necessary to divide the cell membrane or the like finely or completely decompose it, and the cell membrane or the like may be at least locally cleaved or opened so that the outside and inside of the cell communicate with each other. Therefore, the preparation of a microalgae disintegration product does not require severe reaction conditions such as saccharification of wood cellulose (for example, heating at about 180 to 300 ° C.), and does not require decomposition / decomposition of organic substances such as amino acids and organic lipids. Alteration can be avoided.

  The polysaccharides that make up the cell membrane and cell wall of microalgae are rich in diversity, and contain various components such as mannan, xylan, agar, and alginic acid as well as cellulose and hemicellulose. Although the strength of each cell is different, cell disruption can be performed by appropriately selecting from known cell disruption methods. For physical crushing methods, ultrasonic method, freeze-thaw method, osmotic shock method, grinding method using beads or solid powder, method using homogenizer, French press method using shear force by forced extrusion from small holes There is a method of applying gas pressure, and the cell wall and the cell membrane are broken by physical energy such as pressing force / stress distortion, vibration, instantaneous pressure fluctuation, and rapid cell expansion. When stress strain is applied by a grinder or the like in a dry state, the cell wall or the like can be efficiently broken. Microalgae with thin or no cell walls are relatively fragile and can be easily crushed by aeration and stirring of a dispersion of microalgae according to a conventional method. An ultrasonic method, a French press method, a homogenizer method, a grinding method, and the like are suitable for disrupting cells having relatively high strength. Such crushing is performed by, for example, a device for crushing cells with an ultrasonic vibrator commercially available as an ultrasonic sludge reduction device (such as an ultrasonic sludge reduction device manufactured by Nishijima Seisakusho Co., Ltd.) or between disks rotating at high speed. A device for physically damaging the cell wall by supplying a cell dispersion liquid to the gap between the cells and shearing force applied to the disk and crushing (for example, JSCE, Vol.33, No.4, pages 351-359 (2007 )), A device that crushes cells by shear friction generated between beads by high-speed stirring of the cell dispersion introduced into the mill chamber filled with beads using a stirrer equipped with a disk or pin (for example, monthly Earth Environment, Vol.31, No.11, see pages 134-135 (2000)). Chemical crushing methods include enzyme digestion, a method using a surfactant, and a method in which a polysaccharide is hydrolyzed by contacting with a strong acid or a strong base. Alternatively, the protoplasm can be released to the outside of the cell by thermally lysing or decomposing the cell wall, specifically, heating at about 100 to 120 ° C. in an aqueous solution, preferably using an autoclave. By heating at about 100 to 120 ° C. under pressure for about 5 to 15 minutes to cleave the bonds of polysaccharides constituting the cell membrane and so on to soften the cell membrane and the like, the cell membrane and the like are easily broken and the cells collapse. In the thermal cell disruption in the temperature range as described above, inactivation of enzymes contained in the protoplasm and reduction of molecular weight due to hydrolysis of high molecular weight organic compounds such as proteins occur. Decomposition of molecular weight compounds is difficult to proceed. This is an advantage because microorganisms such as yeast do not need to be decomposed using their own enzymes and are easy to use. When the heating temperature is around 200 ° C., low molecular weight compounds such as amino acids are easily decomposed into ammonia, carbon dioxide and the like, so it is important not to give excessive heat.

  Examples of materials that can easily prepare a microalgae disintegration include Chlorella vulgaris, Nannochloropsis, Donariella, Pavlova luteri, etc., and Chlorella vulgaris, Nannochloropsis and Pavlova luteri are cell walls. Is fragile and Donariella does not have a cell wall. Usually, when a physical or chemical disruption treatment as described above is applied to a microalgae, a suspended microalgae collapsed product in which at least a part of the protoplasm is eluted out of the cell is obtained. The outflow of protoplasm can be confirmed by, for example, nitrogen analysis or phosphorus analysis.

  When the crushing process is performed by the method as described above, a microalgae collapse product can be obtained efficiently. The above-mentioned crushing treatment is efficient when the aqueous liquid containing the cultured microalgae in a suspended state is preliminarily concentrated to about 2% by mass to 20% by mass (in terms of dry mass). Concentration of microalgae can be performed using, for example, centrifugation, coagulation sedimentation, filtration, flotation separation and the like. Since seawater is not so concentrated even if it is centrifuged, microalgae cultured in seawater can be well concentrated using a centrifuge or the like. In addition, regarding the influence of seawater salinity on the fermentation system, a salt concentration of about 1% by weight does not adversely affect the fermentation, but in consideration of this point, the microalga culture solution is used so as not to excessively increase the salt concentration of the fermentation system. It is preferable to concentrate and use.

  The protoplasm of microalgae consists of various minerals such as calcium, iron, magnesium, zinc, copper and potassium, and amino acids such as lysine, leucine, isoleucine, phenylalanine, threonine, valine, arginine, alanine, aspartic acid, glutamic acid and glycine. Depending on the type, vitamins such as carotene, vitamins B1, B2, B6, B12, D, E, K1, niacin, folic acid, proteins, lipids, fatty acids, and the like are included. Chlorella and Nannochloropsis provide saturated or unsaturated fatty acids such as palmitic acid, linoleic acid, eicosapentaenoic acid, spirulina provides protein, and Donariella is rich in vitamin A. Therefore, if microalgae disintegration material is supplied to the fermentation system, it becomes a source of essential nutrients and required trace elements of fermentation microorganisms, and cellulose and semi-herulose prepared from biomass waste such as wood waste and rice straw are used as biomass raw materials. Nutrients and elements that are insufficient when the hydrolyzed saccharified product is used as a sugar resource are supplied, and ethanol fermentation can be suitably carried out. The microalgae disintegration product can be used as it is in the fermentation broth, and the cell wall and cell membrane of the microalgae disintegration product do not hinder fermentation. Alternatively, the suspension of the microalgae collapsed product may be pressed or stirred to promote the release of the protoplasm, or the protoplasmic component may be separated from the collapsed product by filtration, extraction or the like. For example, an aqueous liquid containing a protoplasmic component can be obtained by passing an aqueous liquid containing a microalgae disintegrant through a filter having a pore size smaller than the size of algal cells (generally about 4 μm or more). From the viewpoint of work efficiency, the microalgae disintegration product is used as it is by adding it to the fermentation broth, and when the yeast overgrowth during the fermentation and surplus yeast is generated, together with the surplus yeast, the cell wall and cell membrane of the microalgae disintegration product Separation and removal may be performed. Separation and removal can use solid-liquid separation by centrifugation, coagulation sedimentation, or the like.

  The sugar resource for ethanol fermentation is obtained by well-known techniques for preparing hydrolyzed saccharified cellulose and semi-herulose from plant waste. Generally used biomass saccharification techniques include exposure to high temperature and high pressure (for example, temperature of about 200 ° C., pressure of 8 MPa) in the presence of water, hydrolysis using an enzyme, and acid or alkali. There are methods for hydrolysis, and any of them may be used. In the case of wood, lignin may be appropriately removed for saccharification. Industrially, an acid hydrolysis method using sulfuric acid, hydrochloric acid, nitric acid or the like is useful, and a method using sulfuric acid is practical. For example, when hydrolyzing with sulfuric acid, it is hydrolyzed by heating the biomass raw material to 100 to 250 ° C. for about 10 to 100 minutes in an aqueous sulfuric acid solution having a concentration of 1 to 10% by mass to obtain calcium hydroxide and hydroxide. After neutralizing with sodium or the like, the liquid containing the saccharified product can be used as a fermentation raw material.

  Known yeasts can be used as fermenting microorganisms used for ethanol fermentation, such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Brettanomyces coustersii, Sarsina ventricli, Kluyveromyces fragilis, Zymomonas mobilis, Kuru. Examples include Iberomyces marxianus. Moreover, you may utilize the bacterium which introduce | transduced the enzyme gene which has the conversion ability to ethanol by gene recombination. When microorganisms such as Clostridium acetobutylicum, Clostridium beigerinki, Clostridium auran butyricum, Clostridium tetanomorphum are used, alcohol such as butanol and acetone can be produced by fermentation.

  When using a microalgae disruption prepared by physical cell disruption, as a form that can promote nutrient utilization of microorganisms in the fermentation system, aggregating yeast (for example, Hiroshi Tanaka et al., “Development of a highly efficient bioethanol production reactor”, IHI technique, Vol.50, No.1 (2010), see pages 11-17). Aggregating yeast grows with cells adhering to form aggregates of yeast and microbial groups, so the distance between the microorganisms is short, and the yeast is the source of nutrients for the degradation products of the nearby microbial groups. Easy to use as. Highly water-insoluble polymer compounds such as proteins contained in the protoplasts of microalgae that have been disrupted by physical cell disruption can be used after being degraded and reduced in molecular weight by hydrolytic enzymes possessed by fermentation microorganisms or other microorganisms. Therefore, the use of flocculent yeast is advantageous in improving fermentation efficiency because the yeast can easily use amino acids and the like decomposed by hydrolyzing enzymes of nearby microorganisms as nutrient sources. Moreover, since sedimentation is good, it is convenient also in solid-liquid separation after fermentation.

  As a fermentation stock solution, a saccharified product obtained from a biomass raw material and an aqueous solution obtained by adding the aforementioned microalgae collapsed product to water are prepared, and biomass ethanol is produced by inoculating and fermenting the fermenting microorganism. The stock solution is prepared so that the saccharified product (glucose) concentration is about 1 to 20% by mass, preferably about 10% by mass, and the amount of microalgae disintegration added (in terms of dry matter) depends on the type of fermenting microorganism. It is preferable to adjust accordingly, but the stock solution may be adjusted so that the concentration is generally about 0.1 to 1% by mass, preferably about 0.2 to 0.5% by mass. When separating and extracting the protoplasm from the microalgae disintegration product, a liquid containing the protoplasm obtained by removing the cell wall or the like from the microalgae disintegration in an amount corresponding to the above ratio may be used.

  The initial pH of the stock solution is adjusted to about 2.5 to 5.5, preferably about 3.0 to 5.5, inoculated with fermenting microorganisms at a rate of about 1 to 30 g / L, and the temperature is about 30 to 37 ° C. The fermentation proceeds by holding for about 2 to 48 hours. The stock solution in which the microalgae disintegrated material is blended does not need to be supplemented with nutrients and the like, and the fermentation can proceed well without using yeast extract or polypeptone. For example, ethanol can be produced at a rate of about 20 to 25 g-ethanol / (L · h) using a stock solution having a glucose concentration of about 10% by mass.

  In the obtained fermentation broth, ethanol is recovered by distilling the filtrate after removing the solid dispersion by filtration or the like according to a conventional method. Distilled residue after ethanol recovery contains residual sugars such as glucose, organic matter, inorganic salts, etc., and since it is in a sterile state by heating during distillation, it is suitably used as a culture solution for microalgae. it can. Therefore, microalgae such as chlorella grown in fresh water may be inoculated into the distillation residue and cultured, and the proliferated microalgae may be used as a nutrient source for a new fermentation stock solution. Alternatively, the distillation residue may be concentrated and added to a marine microalga culture or fermentation broth. Further, after fermentation, the solid dispersion may be distilled as it is without removing the solid dispersion from the fermentation liquid. In this case, the distillation residual liquid contains fermentation microorganisms and cell membranes of microalgae, but these are separated by filtration or the like. Then, when saccharification treatment is performed together with plant biomass which is a saccharified raw material, it is easily hydrolyzed and can be used as a sugar resource. When separating and extracting protoplasms from microalgae breakdown products or when separating solid products from fermentation broth, the solid matter such as cell walls of microalgae removed is a saccharification process for plant biomass. Should be introduced.

<Ethanol fermentation using chlorella>
(Saccharification of wood waste)
A 1 kg piece of wood from building waste was cut into fine pieces, 1000 ml of a 2% by weight aqueous sodium hydroxide solution was added, and the mixture was kept at 100 ° C. for 30 minutes, and removed by decomposing and eluting lignin to prepare kraft pulp. The pulp was washed with glass fiber filter paper having a pore size of 0.1 μm and 5 L of distilled water, and then dried at 105 ° C. To this pulp, 1000 ml of dilute sulfuric acid having a sulfuric acid concentration of 1.5% by mass was added and heated to 200 ° C. under a pressure of 8 MPa to saccharify, and then a slightly larger molar amount of calcium carbonate than the added sulfuric acid was added. Was removed as a precipitate and used as a raw material saccharified product for the following ethanol fermentation.

(Chlorella culture and disintegration preparation 1)
A part of the saccharified product is dissolved in tap water to prepare 1 L of an aqueous solution having a glucose concentration of 1% by mass, 0.2 g of KNO ₃ , 0.2 g of CaCl ₂ , 0.3 g of MgSO _4. , 0.4 g of citric acid and 0.1 g of iron citrate were added to prepare a culture solution, and 4 g of chlorella (manufactured by Chlorella Kogyo Co., Ltd., raw chlorella V12) (in terms of dry mass) was inoculated under dark conditions at 20 ° C. Cultured for 1 day. This culture solution was concentrated to about 10% by mass (in terms of dry mass) with a centrifuge, and then heated to 120 ° C. for 10 minutes in an autoclave. The obtained aqueous suspension was filtered through a glass filter having a pore size of 0.1 μm and the concentrations of phosphorus and nitrogen were measured. The filtrate had a phosphorus concentration of 100 mg-P / L or more and a nitrogen concentration of 500 mg-N. / L or more, and the aqueous suspension after heating was a dispersion aqueous solution of a chlorella disintegrant in which elution of protoplasm was evident.

(Ethanol fermentation and ethanol recovery)
The saccharified product and chlorella disintegrant dispersion are weighed and added to 1 L of distilled water so that the concentration of saccharified product (glucose) is about 10% by mass and the concentration of chlorella disintegrated product is about 0.5% by mass. The pH was adjusted to about 5.0 to 5.5 using sodium hydroxide. Using this as a fermentation stock solution, 5 g of yeast (aggregating yeast Saccharomyces cerevisiae NCYC1119) was inoculated at a dry mass, and subjected to anaerobic fermentation at about 25 ° C. for about 48 hours.

  Ethanol contained in the obtained fermentation broth was rectified and condensed using a distiller to obtain 35 g of ethanol.

(Chlorella culture and disintegration preparation 2)
10g of distillation residue was recovered from the distiller, and 0.99L of distilled water was added to make a culture solution. 4g of chlorella (Chlorella Kogyo Co., Ltd., raw chlorella-V12) was inoculated and converted to room temperature at 20 ° C. Cultured for 10 days. This culture solution was concentrated about 20 times with a centrifuge, and the chlorella dispersion, which was about 10% by mass (in terms of dry mass), was heated to 120 ° C. for 10 minutes in an autoclave to obtain a chlorella disintegrated product. Using this chlorella decay product, ethanol fermentation and distillation were performed in the same manner as described above, and 38 g of ethanol was obtained.

<Ethanol fermentation using Nannochloropsis>
(Nannochloropsis culture and preparation of disintegration)
Seawater was collected, and biological removal and sterilization treatment in seawater was performed by filtration through a filter having a pore diameter of 0.1 μm and irradiation with ultraviolet rays.

To 1 L of this seawater, add 1 g KNO ₃ , 0.2 g K ₂ HPO ₄ , 0.2 g CaCl ₂ , 0.3 g MgSO ₄ , 0.4 g citric acid and 0.1 g iron citrate. As a culture solution, 1 g of Nannochloropsis (Chlorella Kogyo, refrigerated Nanno Yanmarine K-1) (in terms of dry mass) was inoculated and cultured at about 20 ° C. for 20 days under room lighting. The culture broth was concentrated about 10 times with a centrifuge, and the Nannochloropsis dispersion, which was about 10% by mass (in terms of dry mass), was heated to 120 ° C. for 10 minutes in an autoclave. When this aqueous suspension was filtered through a glass filter having a pore size of 0.1 μm and the concentrations of phosphorus and nitrogen were measured, the filtrate had a phosphorus concentration of 100 mg-P / L or more and a nitrogen concentration of 500 mg-N / L. L was greater than or equal to L, and elution of the protoplasm upon heating was apparent.

(Ethanol fermentation and ethanol recovery)
The saccharified product prepared in Example 1 and the nannochloro prepared according to the above so that the saccharified product (glucose) concentration was 10% by mass and the concentration of the nannochloropsis disintegrated product (in terms of dry mass) was about 0.5% by mass. The psis disintegrant dispersion was weighed and added to 1 L of distilled water, and the pH was adjusted to about 5.0 to 5.5 using hydrochloric acid and sodium hydroxide. Using this as a fermentation stock solution, 5 g of yeast (aggregating yeast Saccharomyces cerevisiae NCYC1119) was inoculated at a dry mass and subjected to anaerobic fermentation at about 25 ° C. for about 48 hours.

  Ethanol contained in the obtained fermentation broth was rectified and condensed using a distiller to obtain 35 g of ethanol.

(Nannochloropsis culture and preparation of disintegration 2)
1000 g of distillation residue was recovered from the distiller, filtered through a filter having a pore size of 0.1 μm, and separated into 50 g (dry mass) of the residue on the filter and 950 g of filtrate. The residue on the filter, 50 g, was used to prepare a new saccharified product in addition to the wood waste.

  Water from the filtrate was distilled off and concentrated, and 1 L of seawater after biological removal and sterilization was added to make a culture solution, inoculated with 1 g of Nannochloropsis (Chlorella Kogyo, refrigerated Nanno Yanmarine K-1), and indoor lighting When cultured for 40 hours under the condition, Nannochloropsis was able to grow to about 2 g (in terms of dry mass). The culture broth was similarly concentrated in a centrifuge and heated to 120 ° C. for 10 minutes in an autoclave to obtain a nonnochloropsis disintegrant. Using this Nannochloropsis decay product, ethanol fermentation and ethanol recovery were possible as described above, and 34 g of ethanol was obtained.

<Ethanol fermentation of rice straw>
The ground rice straw was heated with superheated steam at 200 ° C. for 3 minutes to saccharify hemicellulose, and the residue was washed with water to obtain kraft pulp. Distilled water was added so that the kraft pulp was 10% by dry weight, and cellulase was added so as to be 5% by weight and kept at 50 ° C. for 48 hours to obtain a saccharified product. Using this saccharified product, as in Example 1, a fermentation stock solution to which a chlorella disintegrated product was added was prepared, inoculated with yeast, and fermented. 39 g of ethanol was obtained by distillation of the obtained fermentation broth.

(Comparative Example 1)
A fermentation stock solution was prepared in the same manner as in Example 2 except that the Nannochloropsis disintegrated product was not used. When fermentation and distillation were carried out, the obtained ethanol was 20 g.

(Comparative Example 2)
A fermentation stock solution was prepared in the same manner as in Example 3 except that the chlorella disintegrated material was not used. After fermentation and distillation, the obtained ethanol was 16 g.

  The present invention provides an economical and rational supply of nutrients and trace elements necessary for fermenting microorganisms by utilizing resources that do not have a risk of a rise in food prices when producing biomass ethanol using plant waste as biomass. Since it contributes to waste disposal and resource production, it is highly useful and can contribute to promotion of recycling and environmental protection.

Claims (10)

  By preparing a saccharified product of a plant polysaccharide and a microalgae disintegrated product whose protoplasm is released to the outside of the cell, and subjecting the saccharified product to ethanol fermentation using a microorganism in the presence of the microalgae disintegrated product, A method for producing ethanol, characterized in that a protoplasm released from the microalgae collapsed material can be used as a source of essential nutrients of microorganisms or required trace elements.   2. The method for producing ethanol according to claim 1, wherein the microalgae collapse product is a collapsed product of at least one microalgae selected from the group consisting of chlorella, Nannochloropsis, Botriococcus and Donariella.   The method for producing ethanol according to claim 1 or 2, wherein the microalgae collapsed product is obtained by heating microalgae in an aqueous liquid.   The method for producing ethanol according to claim 1 or 2, wherein the microalgae collapsed product is obtained by crushing a cell membrane of microalgae.   The method for producing ethanol according to any one of claims 1 to 4, wherein the vegetable polysaccharide contains cellulose or hemicellulose, and the saccharified product is a monosaccharide containing glucose.   The method for producing ethanol according to any one of claims 1 to 5, wherein the saccharified product is a hydrolyzate of plant polysaccharides with an alkali or acid, or an enzyme-decomposed product with cellulase.   The microorganism comprises at least one fermenting microorganism selected from the group consisting of bacteria and yeast, and the protoplasm released from the microalgae collapse contains essential nutrients and required trace elements of the fermenting microorganism. Item 7. The method for producing ethanol according to any one of Items 1 to 6.   The method for producing ethanol according to any one of claims 1 to 7, wherein the microorganism comprises an aggregating yeast.   The ethanol fermentation is a step of preparing an aqueous liquid having a saccharified concentration of 1 to 20% by mass and a content of microalgae disintegrant (in terms of dry matter) of 0.1 to 1% by mass, and adjusting the pH of the aqueous liquid. A step of adjusting to 2.5 to 5.5, a step of inoculating the aqueous microorganism with the microorganism, and a step of maintaining the aqueous liquid after the inoculation at a temperature of about 30 to 37 ° C. for 2 to 48 hours. Item 9. A method for producing ethanol according to any one of Items 1 to 8.   The manufacturing method of ethanol in any one of Claims 1-9 which culture | cultivate a micro algae using the residue after isolate | separating ethanol from the product of the said ethanol fermentation.