JP2012050376A - Method for producing ethanol - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing ethanol that can effectively produce the ethanol while suppressing proliferation or action of bacteria.SOLUTION: There is provided the method for producing ethanol including a process for culturing microorganisms capable of performing ethanol fermentation in the presence of ethanol having concentration enough to suppress the action of the bacteria wherein the ethanol having the concentration enough to suppress the action of the bacteria exists from the start of the culture. In one embodiment, the method further includes a process for continuously or intermittently recovering the ethanol.

Description

  The present invention relates to a method for producing ethanol.

  Production of useful substances such as ethanol, amino acids, and organic acids using microorganisms is widely and industrially performed at home and abroad. In particular, bioethanol using biomass, which is a renewable resource, has attracted attention from the viewpoint of preserving fossil resources and preventing global warming. Bioethanol production from sugarcane and corn has already been put into practical use and used by mixing it with gasoline. However, since these raw materials are also food, it has been pointed out that the widespread use of bioethanol leads to soaring food and food crisis in developing countries. For this reason, in recent years, research and development has been promoted using non-edible parts of plants such as rice straw, corn stover, bagasse and wood. In Japan, some ethanol production plants that use rice straw and waste building materials as raw materials have begun to operate, but there remains a big problem of high cost.

  Japan is a country surrounded by greenery, but it is actually poor in biomass, and it is pointed out that even in the biomass Japan comprehensive strategy, even if ethanol is produced from all biomass available in Japan, it is only 6 million kL. Yes. For this reason, the development of new biomass is required, and technological development to produce ethanol from biomass that is difficult to handle due to effective use of waste such as waste cotton fiber and waste paper, and food waste is likely to rot. It is being advanced.

  Japanese biomass has a low yield per unit area and is a small variety of varieties except rice straw. To minimize the energy required to transport bulky and high-moisture biomass and improve production efficiency from individual biomass, even small-scale production that shortens the transportation distance of raw materials consumes less energy and is less expensive Development of production system is necessary. Furthermore, conventional ethanol production is liquid fermentation in which water accounts for 80% or more of the system. In conventional distillation methods, the temperature of the fermentation broth including this large amount of water is manipulated, so a great deal of energy is required to recover ethanol, and after that a great deal of energy and energy is required to dispose of a large amount of waste water and high-moisture residues. Spend the cost. In addition, most of the residue is incinerated, and potassium phosphate and other trace elements contained therein are not reduced to farmland.

  On the other hand, the present inventors have proposed a Consolidated Continuous Solid State Fermentation (CCSSF) system as an ethanol production system (Non-patent Document 1).

  In the production of bioethanol, various techniques have been developed as described above, but the problem that still remains in actual production is the growth of various bacteria during fermentation. Fermentation with sugar such as sugar cane or sugar obtained by hydrolysis of starch or cellulose with microorganisms such as yeast will yield ethanol. The sugar is metabolized to a substance other than ethanol such as lactic acid to reduce the yield of ethanol, and in some cases, ethanol fermentation by yeast itself is inhibited (Non-patent Documents 2 and 3).

  To avoid this, the raw material is heat sterilized and fermented in an aseptic environment. Antibiotics that suppress the growth of various bacteria and antibacterial agents such as potassium disulfite are added. Countermeasures such as ethanol fermentation are attempted (Non-Patent Documents 4 to 6). For example, Non-Patent Document 4 describes a method of using lactate-resistant yeast, Candida glabrata, to add lactate to a medium in order to prevent contamination with lactic acid bacteria in bioethanol production. . Non-patent document 5 describes a method of adding acetate (acetate) to a culture medium in bioethanol production and using acetic acid-resistant yeast, Schizosaccharomyces pombe. . Non-Patent Document 6 describes an attempt to add the antibiotic virginiamycin to the medium in order to prevent bacterial contamination and infection in bioethanol production.

  However, heat sterilization of a large amount of biomass requires a lot of energy, and the addition of antibiotics and antibacterial agents is not only costly but also has a problem of polluting the environment. Yeast that can maintain its strength has not been developed yet.

  When rice straw and waste building materials are used as raw materials, pre-treatment for saccharification involves delignification using heating with dilute sulfuric acid and subcritical water at 160 ° C or higher. There is a problem that various bacteria contained in the saccharifying enzyme to be added and various bacteria mixed during fermentation grow. In particular, when food waste, waste cotton fiber, and waste paper that do not require delignification treatment are used as raw materials, the problem that ethanol yield is significantly reduced unless measures are taken against miscellaneous germs derived from raw materials. It is in the state of.

Moukamnerd, C. et al., Appl. Microbiol. Biotechnol., (2010). DOI 10.1007 / s00253-010-2716-y Muthaiyan, A. and Ricke, C. S. Bioresour. Technol., 101, 5033-5042 (2010) Schell, D. J. et al., Bioresour. Technol., 98, 2942-2948 (2007) Watanabe, I. et al., J. Ind. Microbiol. Biotechnol., 35, 1117-1122 (2008) Saithong, P. et al., J. Biosci. Bioeng., 3, 216-219 (2009) Bischoff, K. M. et al., Biotechnol. Bioeng., 103, 117-122 (2009)

  The objective of this invention is providing the manufacturing method of ethanol which can produce ethanol efficiently, suppressing the proliferation and activity of various bacteria.

The present invention provides a method for producing ethanol, which comprises:
Culturing a microorganism capable of ethanol fermentation in the presence of a concentration of ethanol capable of suppressing the activity of various bacteria,
A concentration of ethanol that can suppress the activity of the bacteria is present from the beginning of the culture.

  In one embodiment, the method further comprises the step of recovering ethanol continuously or intermittently.

The present invention also provides a method of suppressing miscellaneous bacteria in ethanol fermentation by a microorganism,
A step of culturing a microorganism capable of ethanol fermentation in the presence of a concentration of ethanol capable of suppressing the activity of various bacteria is included.

  ADVANTAGE OF THE INVENTION According to this invention, the manufacturing method of ethanol which can produce ethanol efficiently is provided, suppressing the proliferation and activity of various bacteria.

1 is a schematic diagram illustrating one embodiment of a CCSSF system. It is the graph which plotted the logarithm of the value which remove | divided the number of living bacteria after 4.5-hour culture | cultivation of yeast and lactic acid bacteria by the number of initial living bacteria with respect to initial stage ethanol concentration. Changes in ethanol concentration and lactic acid concentration in the reaction tank over time (upper) and changes in the number of viable yeast and lactic acid bacteria over time (lower) in CCCSF when 700 units of glucoamylase and α-amylase were added without adding ethanol. ). Changes in the ethanol concentration and lactic acid concentration in the reaction tank over time (upper) and changes in the number of viable yeast and lactic acid bacteria over time (lower) in CCSSF when 300 units of glucoamylase and α-amylase were added without adding ethanol. ). It is a graph which shows the time-dependent change (upper) of the ethanol concentration and lactic acid concentration in a reaction tank in CCCSF at the time of adding ethanol beforehand, and the time-dependent change (lower) of the viable count of yeast and lactic acid bacteria.

  The present invention is based on the finding that the activity or growth of various germs can be suppressed by culturing a microorganism in the presence of ethanol in ethanol fermentation by the microorganism.

  The present invention provides a method for producing ethanol, the method comprising the step of culturing a microorganism capable of ethanol fermentation in the presence of a concentration of ethanol capable of suppressing the activity of various bacteria. A concentration of ethanol that can suppress the activity of the bacteria is present from the beginning of the culture.

  The ethanol production method of the present invention is characterized in that ethanol at a concentration capable of suppressing the activity of various bacteria is present when ethanol fermentation by microorganisms is started.

  Microorganisms used in the present invention need only have ethanol fermentation ability, but are resistant to ethanol, such as yeasts such as Saccharomyces and Schizosaccharomyces, and bacteria such as Zymomonas. It is desirable to have a microorganism. It is desirable that the microorganism is resistant to ethanol, that is, a microorganism that can maintain a high ethanol production amount per hour per cell even in the presence of a high concentration of ethanol.

  Specifically, Saccharomyces cerevisie, especially yeast such as sake yeast, brewer's yeast, wine yeast, and baker's yeast (preferably used for brewing alcoholic beverages with high alcohol content such as shochu, sake, whiskey, etc. Yeast), bacteria such as Zymomonas mobilis, these microorganisms acclimated to ethanol, classical microorganisms such as mutation treatment, and those microorganisms whose ethanol tolerance has been enhanced by genetic recombination. Moreover, microorganisms imparted with ethanol fermentation ability by genetic recombination, for example, Escherichia coli or Corynebacterium bacteria into which a gene of a pathway from pyruvic acid to ethanol is incorporated can also be used.

  A fermentation raw material can be used for culturing a microorganism capable of ethanol fermentation. The fermentation raw material may be any raw material that can be used for fermentation to obtain ethanol, and includes carbohydrates such as glucose, sucrose, maltose, xylose, galactose, mannose, starch, cellulose, hemicellulose, etc. As such, it can be a substance other than a carbohydrate. Moreover, a fermentation raw material is not limited to any of these, A multiple types of mixture may be sufficient.

  When fermentation raw materials contain high-molecular carbohydrates such as starch, cellulose, and hemicellulose, sugars (monosaccharides, disaccharides, etc.) that allow microorganisms to ferment ethanol with enzymes, acids, alkalis, subcritical water, supercritical water, etc. ) Can be hydrolyzed (saccharified), followed by ethanol fermentation to the microorganism. The enzyme for saccharification (saccharifying enzyme) depends on the type of raw material, and examples thereof include amylase, cellulase, hemicellulase and the like, and these may be used in combination as necessary. These saccharifying enzymes include various types of enzymes by cleaving their substrates (amylase includes α-amylase, β-amylase, glucoamylase, isoamylase, etc., and cellulase includes endoglucanase, cellobio) Hydrolase, β-glucanase, etc.) may be used in combination as necessary. Alternatively, a saccharification enzyme and a microorganism capable of ethanol fermentation can be added simultaneously to a fermentation raw material containing a high molecular weight carbohydrate, and ethanol fermentation by a microorganism using a sugar produced by saccharification of the carbohydrate and saccharification can be performed in parallel (this can be done). It is also called “parallel double fermentation”).

  The fermentation raw material may be an agricultural, industrial, or food waste including starch, cellulose, hemicellulose, lignocellulose, and the like. When using lignocellulose or waste such as rice straw and waste building materials containing lignocellulose as a raw material, delignification treatment can be performed with subcritical water or dilute sulfuric acid as a pretreatment for saccharification. Food wastes including starch, cellulose, hemicellulose, and wastes such as waste cotton fibers and waste paper can also be pretreated if necessary and used for saccharification and fermentation.

  The concentration of ethanol present during ethanol fermentation by microbial culture is a concentration that can suppress the activity of various bacteria, and is preferably a concentration that does not inhibit ethanol fermentation by microorganisms capable of ethanol fermentation.

  Examples of miscellaneous bacteria that can be mixed during ethanol fermentation include lactic acid bacteria of the genus Lactobacillus, such as Lactobacillus plantarum, Lactobacillus paracasei, and Lactobacillus fermentum.

  The appropriate ethanol concentration may vary depending on factors such as the type of microorganisms that can be ethanol-fermented, the type and concentration of various germs to be mixed, the type of fermentation material, the concentration of antimicrobial components contained in the fermentation material, and the fermentation system. For example, when using raw materials in which various bacteria have already grown to a considerable concentration, or when the mixed bacteria are microorganisms having a relatively high resistance to ethanol, they are relatively high at around 50 g / kg at the start of fermentation. It is desirable to have a concentration of ethanol present. Conversely, when the concentration of miscellaneous bacteria present at the start of fermentation is low, or when ethanol contamination of the contaminated miscellaneous bacteria is low, the concentration of ethanol present at the start of fermentation can be about 20 to 30 g / kg.

  Also in the following cases, the concentration of ethanol present at the start of fermentation can be set low. For example, when lignocellulose is used as a raw material, delignification treatment is performed with subcritical water or dilute sulfuric acid as a pretreatment, and furfurals and phenolic compounds produced at this time are included in the raw material, and these are miscellaneous bacteria. Can suppress the activity. In addition, when food-based waste is used as a raw material, the original food contains preservatives and antibacterial components (for example, antibacterial components derived from wasabi, mustard, pepper etc., antibacterial components such as chitosan and catechin), etc. There may be. In such a case, since these substances contained in the raw material and ethanol can act synergistically to suppress germs, even if the concentration of ethanol present is set low, a substantial germ suppression effect can be obtained.

  The ethanol concentration after the start of fermentation depends on the above factors, but may be 20 g / kg or more, preferably 40 g / kg or more, more preferably 50 g / kg or more. The presence of as high a concentration of ethanol as possible can reliably suppress the activity of bacteria, but from the viewpoint of maintaining the fermentative power of microorganisms capable of ethanol fermentation, It is desirable to maintain it in a range not exceeding / kg.

  The timing and method of adding ethanol are not particularly limited as long as an appropriate amount of ethanol is present in the fermentation mixture at the start of culturing microorganisms for ethanol fermentation. Prepare a fermentation mixture containing microorganisms that can be ethanol-fermented and fermentation raw materials (optionally secondary ingredients containing saccharifying enzymes) (e.g., in the tank, microorganisms, fermentation raw materials, and, if necessary) for fermentation Can be added at the time of charging a secondary material containing saccharifying enzyme. If it is a raw material which is easy to rot, it can be added to the raw material at the time when the raw material is obtained, and the proliferation of germs until the start of fermentation can be suppressed. For example, in the case of starch-based waste discharged from a food factory, the activity of germs during storage and transportation can be suppressed by adding an appropriate amount of ethanol when discharged as waste. In this case, the amount of ethanol added can suppress the activity of miscellaneous bacteria when mixed with microorganisms that can be ethanol-fermented and, if necessary, auxiliary materials (preferably microorganisms that can be ethanol-fermented). (The fermentative power of can also be maintained). In the case of raw materials that are easily contaminated by various germs, or raw materials that are already contaminated by various germs, for example, ethanol is added to a concentration of 100 g / kg or more. The ethanol concentration at the start of fermentation may be set to a predetermined concentration by mixing with another raw material that has not been used. Examples of the addition method include adding ethanol to the fermentation mixture so as to obtain a predetermined concentration, and immersing the fermentation raw material in an ethanol solution having a predetermined concentration.

  The concentration of the ethanol itself to be added is not particularly limited, and may be an appropriate concentration at the time of starting the culture of microorganisms for ethanol fermentation, and the origin and purity of the added ethanol are not particularly limited. Chemically synthesized ethanol, ethanol obtained by brewing, etc. can also be used, but from the viewpoint of cost, ethanol produced by the ethanol production method, alcoholic beverages discarded for reasons such as quality and expiration date, etc. Can be suitably used.

  Conditions suitable for culturing microorganisms capable of ethanol fermentation can be conditions suitable for culturing microorganisms to be used. The ethanol fermentation process to which the present invention is applied may be so-called solid fermentation (for example, CCSSF described in Non-Patent Document 1) having a moisture content of 30 to 80%, and a moisture content of 80 to 90% or more. It may be liquid fermentation. A medium for culturing a microorganism capable of ethanol fermentation includes a fermentation raw material and, optionally, a secondary raw material including a saccharifying enzyme, and may further include components normally used for culturing a microorganism.

  The ethanol fermentation process is desirably a process that continuously or intermittently recovers ethanol during fermentation (eg, CCSSF), but may be a normal production process that does not recover ethanol during fermentation. If ethanol is not recovered during fermentation, the final concentration of ethanol when fermenting a certain amount of sugar increases by the amount of ethanol present at the start of fermentation, which tends to lead to delays in fermentation and a decrease in yield. This does not prevent the application of the present invention.

  As an ethanol fermentation process to which the present invention is applied, CCCSF can be preferably used. FIG. 1 is a schematic diagram illustrating one embodiment of a CCSSF system. The CCSSF system 10 includes a drum-type reaction tank 11 (including the fermentation mixture 12), a warm water tank 13, a condensation tower 14 (collecting ethanol 15), a humidification tank 16, and a pump 17. The drum-type reaction vessel 11 is rotatable and can be used for fermentation with an internal fermentation mixture 12 (including a microorganism capable of ethanol fermentation and a fermentation raw material, and optionally a secondary raw material containing a saccharifying enzyme). The drum-type reaction tank 11 can be connected to the condensation tower 14 and the humidification tank 16 so that the gas in the head space can be circulated. The insulated water tank 13 can contain a liquid (for example, water) at a temperature (for example, 37 ° C.) that can maintain the inside of the reaction tank at a temperature suitable for fermentation. The condensing tower 14 includes a refrigerant circulation path and an ethanol recovery container, and condenses the ethanol gas discharged from the drum-type reaction tank 11 by circulating a refrigerant (for example, water added with a commercial antifreeze) using the path. The generated liquid ethanol 15 can be recovered in the lower ethanol recovery container. The pump 17 may be provided between the condensation tower 14 and the humidification tank 16 in order to circulate the head space gas of the drum type reaction tank 11 to the condensation tower 14 and the humidification tank 16. Moisture in the fermentation mixture will evaporate with the ethanol into the headspace, thereby reducing the moisture in the fermentation mixture, which can be compensated for by the humidifier 16. In the CCSSF system 10, the ethanol concentration in the fermentation mixture can be adjusted by adjusting the circulation rate of the head space gas from the drum reactor 11 to the condenser tower 14. Moreover, the water | moisture content in a fermentation mixture can be adjusted by adjusting the temperature of the humidification tank 16. FIG.

  In the CCSSF system, ethanol is produced in a reaction tank by parallel double fermentation in which microorganisms capable of ethanol fermentation, fermentation raw materials (biomass pretreated as necessary), and saccharifying enzyme are reacted in the presence of a minimum amount of water. When the ethanol in the reaction tank reaches an appropriate concentration, the headspace gas can be circulated through the condensing tower to start the ethanol recovery. In the condensing tower, the ethanol gas discharged from the reaction tank can be condensed to recover the ethanol as a liquid. In the CCSSF system, by adjusting the circulation rate of the gas in the head space in accordance with ethanol production by microorganisms in the fermenter, ethanol can be recovered while maintaining a constant ethanol concentration in the reaction vessel.

  This CCSSF system is simple and compact because saccharification, fermentation, and ethanol recovery are performed in one system. In addition, no waste water is produced and the residue can be reused as compost by simple post-treatment. Therefore, the CCSSF system can realize energy saving and low cost while being a small-scale system that minimizes the transport distance of raw materials.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated, this invention is not limited by this Example.

  A lactic acid bacterium (Lactobacillus plantarum NRIC1067) was selected as a model of miscellaneous bacteria mixed in ethanol fermentation, and left to stand overnight at 37 ° C. in MRS medium (Difco, Becton Dickinson). Moreover, baker's yeast (Saccharomyces cerevisiae) was used as a microorganism for ethanol fermentation.

(Reference example: Effect of ethanol on the growth of lactic acid bacteria and yeast)
7 × 10 ⁶ CFU (colony forming unit) lactic acid bacteria or 7 × 10 ⁸ CFU yeast in 4% glucose and various concentrations (0, 20, 40, or 60 g / kg) of YP medium (1% Yeast extract, 2% peptone, pH 6.0) (both Difco and Becton Dickinson) were inoculated into a culture medium containing 3.9 ml and corn starch 5 g, and cultured at 37 ° C. for 4.5 hours. The viable numbers of lactic acid bacteria and yeast before (initial) and after 4.5 hours of culture were measured by a plate culture method using an MRS agar medium and a YPD agar medium, respectively.

  FIG. 2 is a graph plotting the logarithm of the value obtained by dividing the number of viable cells after yeast and lactic acid bacteria were cultured for 4.5 hours by the number of viable initial cells against the initial ethanol concentration. The vertical axis indicates the logarithm of the value obtained by dividing the number of viable cells after 4.5 hours of culture by the initial viable cell number, and the horizontal axis indicates the ethanol concentration (g / kg) at the start of the culture. White triangles represent lactic acid bacteria, and black triangles represent yeast. When the logarithm is a positive value, it indicates an increase in the number of viable bacteria, and when it is a negative value, it indicates a decrease.

  When the ethanol concentration at the start of the culture was 0 and 20 g / kg, the number of viable lactic acid bacteria increased, but at 40 g / kg, there was almost no increase, and at 60 g / kg, the viable cell count decreased. On the other hand, the viable cell count of yeast was almost constant up to 40 g / kg, although it slightly decreased when the ethanol concentration at the start of the culture was 60 g / kg.

  These results indicate that the growth of lactic acid bacteria can be suppressed while maintaining the fermentability of yeast, if the ethanol concentration at the start of culture is 20 to 60 g / kg, preferably 40 to 60 g / kg. . At 20 g / kg, growth of lactic acid bacteria is observed, but there is no problem.

(Construction of CCSSF system)
The CCSSF system shown in FIG. 1 was constructed. The CCSSF system 10 includes a drum-type reaction tank 11 (including the fermentation mixture 12), a warm water tank 13, a condensation tower 14 (collecting ethanol 15), a humidification tank 16, and a pump 17. The drum-type reaction tank 11 has a diameter of 10 cm and a length of 15 cm, is rotatable, and has a fermentation mixture 12 provided for fermentation therein. The drum-type reaction tank was connected to the agglomeration tower 14 and the humidification tank 16 so that the gas in the head space could be circulated. In order to maintain the temperature inside the reaction tank 11, 37 ° C. water was added to the heat insulation water tank 13. The condensing tower 14 is provided with a refrigerant circulation path and an ethanol recovery container. As the refrigerant, water added with commercial antifreeze is used to condense the ethanol gas discharged from the drum-type reaction tank 11, and the resulting liquid ethanol 15 is recovered. It collected in the ethanol collection container. A pump 17 was provided between the condensing tower 14 and the humidifying tank 16, and the head space gas in the drum type reaction tank 11 was circulated to the condensing tower 14 and the humidifying tank 16. The humidification tank 16 compensated for a decrease in the water content of the fermentation mixture caused by evaporation of the water content in the fermentation mixture into the headspace together with ethanol. The ethanol concentration in the fermentation mixture is adjusted by adjusting the circulation speed of the head space gas from the drum-type reaction tank 11 to the condensing tower 14, and the moisture in the fermentation mixture is adjusted by adjusting the temperature of the humidification tank 16. did.

(Comparative Example 1)
Preparation of fermentation mixture 12 containing corn starch 50 g, YP medium 65 mL, yeast 30 g (corresponding to 6 g as dry weight: 1 × 10 ⁸ cfu / g), and 700 units each of glucoamylase and α-amylase as saccharifying enzymes Ten drum-type reaction tanks 11 were placed. At the same time, lactic acid bacteria were also added into the drum-type reaction tank 11 so as to be 1 × 10 ⁶ cfu / g. The drum-type reaction tank 11 is placed in a warm water tank 13 containing water at 37 ° C., rotated at 5 rpm, and subjected to ethanol fermentation by the CCSSF system. Ethanol and lactic acid concentrations in the reaction tank 11, lactic acid bacteria and yeast live bacteria The number was monitored. The concentration of ethanol in the reaction tank 11 and the concentration of ethanol 15 recovered in the agglomeration tower 14 were measured by an enzyme electrode method. The lactic acid concentration in the reaction tank 11 was also measured by the enzyme electrode method. The numbers of living lactic acid bacteria and yeast were measured in the same manner as in the above Reference Example.

  After the ethanol concentration in the reaction tank 11 reaches a predetermined concentration, the headspace gas in the reaction tank 11 is supplied to the condensing tower 14 set to −10 ° C. and the humidification tank 16 set to 47 ° C. to about 0.5 L / Circulation was performed at a flow rate of min, and then the flow rate of the circulation pump was appropriately adjusted so that the ethanol concentration was maintained at a predetermined concentration.

  FIG. 3A shows changes in ethanol concentration and lactic acid concentration in the reaction tank over time (upper) and the viable numbers of yeast and lactic acid bacteria in CCCSF when 700 units of glucoamylase and α-amylase were added without adding ethanol. It is a graph which shows a time-dependent change (lower). In the upper graph of FIG. 3A, the left vertical axis indicates the ethanol concentration (g / kg) in the reaction tank, the right vertical axis indicates the lactic acid concentration (g / kg) in the reaction tank, and the horizontal axis indicates Shows the fermentation time (h), black circles represent ethanol, and white squares represent lactic acid. In the lower graph of FIG. 3A, the vertical axis represents the viable cell count (CFU / g), the horizontal axis represents the fermentation time (h), the black triangle represents yeast, and the white triangle represents lactic acid bacteria.

  The ethanol concentration in the reaction tank reached 50 g / kg about 7 hours after the start of fermentation. Thereafter, the ethanol concentration in the reaction vessel was maintained at 50 g / kg by adjusting the circulation rate. Lactic acid bacteria continued to grow until the 12th hour, and the number of living lactic acid bacteria at the end of the fermentation increased to three times the initial number. Along with this, 1.5 g / kg of lactic acid was produced.

(Comparative Example 2)
In parallel double fermentation including CCSSF, the saccharification process is generally the rate-limiting step, and the slower the saccharification rate, the longer it takes for the ethanol concentration to reach a predetermined concentration. The yield of ethanol is reduced. Therefore, assuming that the saccharification rate is low, the case where the amount of glucoamylase and α-amylase in the fermentation mixture was 300 units was also examined. Except for the amounts of glucoamylase and α-amylase, it was subjected to ethanol fermentation by the CCSSF system in the same manner as in Comparative Example 1 above, and the ethanol concentration and lactic acid concentration in the reaction tank, and the number of living lactic acid bacteria and yeast were monitored.

  FIG. 3B shows changes over time in ethanol concentration and lactic acid concentration in the reaction tank (upper) and the viable numbers of yeast and lactic acid bacteria in CCCSF when 300 units of glucoamylase and α-amylase were added without adding ethanol. It is a graph which shows a time-dependent change (lower). The axes and symbols of each graph in FIG. 3B are similar to each graph in FIG. 3A.

  The ethanol concentration in the reaction tank reached 50 g / kg 12 hours after the start of fermentation. Thereafter, the circulation rate was adjusted to maintain the ethanol concentration at 50 g / kg. Thereafter (after 12 hours from the start of fermentation), no growth of lactic acid bacteria was observed, but the number of live lactic acid bacteria at the end of fermentation increased to 10 times the initial number. Along with this, 2.3 g / kg of lactic acid was produced.

  From the results of Comparative Examples 1 and 2 above, it can be seen that lactic acid bacteria proliferate while the ethanol concentration is low, and when the ethanol concentration reaches a certain concentration, the growth of lactic acid bacteria stops. However, it can be seen that the sugar is converted to lactic acid by the lactic acid bacterium that has grown without fear that the growth of the lactic acid bacterium will stop. This indicates that the activity of lactic acid bacteria becomes more prominent as the ethanol concentration increases more slowly.

Example 1
The content of glucoamylase and α-amylase in the fermentation mixture is 300 units, respectively, and ethanol is added to the fermentation mixture in advance so that the ethanol concentration in the reaction tank at the start of fermentation is 40 g / kg. In the same manner as in Comparative Example 1 above, ethanol fermentation by the CCSSF system was performed, and the ethanol concentration and lactic acid concentration in the reaction tank, and the viable cell counts of lactic acid bacteria and yeast were monitored.

(Example 2)
The content of glucoamylase and α-amylase in the fermentation mixture is 300 units, respectively, and ethanol is added to the fermentation mixture in advance so that the ethanol concentration in the reaction tank at the start of fermentation is 50 g / kg. In the same manner as in Comparative Example 1 above, ethanol fermentation by the CCSSF system was performed, and the ethanol concentration and lactic acid concentration in the reaction tank, and the viable cell counts of lactic acid bacteria and yeast were monitored.

(Example 3)
The content of glucoamylase and α-amylase in the fermentation mixture is 300 units, respectively, and ethanol is added to the fermentation mixture in advance so that the ethanol concentration in the reaction tank at the start of fermentation is 60 g / kg. In the same manner as in Comparative Example 1 above, ethanol fermentation by the CCSSF system was performed, and the ethanol concentration and lactic acid concentration in the reaction tank, and the viable cell counts of lactic acid bacteria and yeast were monitored.

  The results of Examples 1 to 3 are shown in FIG. FIG. 4 is a graph showing changes over time in ethanol concentration and lactic acid concentration in the reaction tank (upper) and changes over time in the number of viable yeast and lactic acid bacteria (lower) in CCSSF when ethanol is added in advance. In the upper graph of FIG. 4, the left vertical axis indicates the ethanol concentration (g / kg) in the reaction tank, the right vertical axis indicates the lactic acid concentration (g / kg) in the reaction tank, and the horizontal axis indicates Fermentation time (h) is shown, black symbols represent ethanol, and open symbols represent lactic acid. In the lower graph of FIG. 4, the vertical axis indicates the viable cell count (CFU / g), the horizontal axis indicates the fermentation time (h), the black symbol indicates yeast, and the white symbol indicates lactic acid bacteria. Represents. In each graph, the square is Example 1 (ethanol concentration in the reaction tank 40 g / kg), the circle is Example 2 (ethanol concentration in the reaction tank 50 g / kg), and the triangle is Example 3 (in the reaction tank). Represents results for ethanol concentration 60 g / kg).

  When the ethanol concentration was maintained at 40 g / kg from the start of fermentation, the number of viable lactic acid bacteria did not increase, and the lactic acid concentration until 18 hours after fermentation was lower than Comparative Examples 1 and 2 above, 0.7 g / kg. It was. When kept at 50 g / kg, the number of viable lactic acid bacteria did not increase, and the lactic acid concentration until 18 hours after fermentation was even lower, 0.2 g / kg. When kept at 60 g / kg, the number of living lactic acid bacteria gradually decreased, and the lactic acid concentration after 18 hours of fermentation also decreased to 0.1 g / kg or less, but the number of viable yeast decreased after 12 hours of fermentation. Although not shown in the figure, the ethanol production rate determined from the change over time in the amount of recovered ethanol decreased.

  Table 1 below shows the ethanol concentrations in the reaction tanks of Comparative Examples 1 and 2 and Examples 1 to 3 (at the start of fermentation and at the steady state), and the saccharifying enzyme concentration (glucose) contained in the fermentation mixture at the start of fermentation. The total amount of amylase and α-amylase), the lactic acid concentration after the end of fermentation, and the ethanol yield are shown.

  The ethanol yield in Table 1 above is (the amount of ethanol recovered (g) + the amount of ethanol in the fermenter (g) −the amount of ethanol added (g)) / (from the starch in the medium at the start of fermentation). Calculated by the calculated glucose amount (g) -residual glucose amount after completion of fermentation (g)).

  The theoretical yield of ethanol from glucose is 0.51 g (ethanol amount) / g (glucose amount) (= 46 × 2/180). In Comparative Examples 1 and 2, lactic acid bacteria grow and consume starch-derived glucose to produce lactic acid and the like, so the ethanol yield is extremely low. In particular, in Comparative Example 2 in which the saccharification is slow and the ethanol concentration is slowly increased, the ethanol yield was 0.34, which was only 2/3 of the theoretical yield. On the other hand, in Examples 1-3, the ethanol yield increased with the ethanol concentration to be added. When 60 g / kg was added, the yield was 0.50 g / g, which was almost equivalent to the theoretical yield.

  In ethanol production by microbial fermentation, if microorganisms are cultured in the presence of ethanol, the growth of miscellaneous bacteria can be suppressed and the yield of ethanol can be kept high. In the case of the CCSSF system, the added ethanol can also be recovered in the condensing column, so there is no cost. A part of the ethanol recovered in the condensing tower can be returned to the reaction tank to perform the next fermentation. When food-based waste that is easily spoiled is used as a raw material for fermentation, an appropriate amount of ethanol is added and transported when waste is generated, and CSSSF can be used to take measures against germs without cost.

DESCRIPTION OF SYMBOLS 10 CCSSF system 11 Drum type reaction tank 12 Fermentation mixture 13 Insulated water tank 14 Condensing tower 15 Ethanol 16 Humidification tank 17 Pump

Claims (3)

A method for producing ethanol, comprising:
Culturing a microorganism capable of ethanol fermentation in the presence of a concentration of ethanol capable of suppressing the activity of various bacteria,
A method wherein a concentration of ethanol capable of suppressing the activity of the bacteria is present from the beginning of the culture.   The method according to claim 1 or 2, further comprising a step of recovering ethanol continuously or intermittently. A method for suppressing miscellaneous bacteria in ethanol fermentation by microorganisms,
A method comprising culturing a microorganism capable of ethanol fermentation in the presence of a concentration of ethanol capable of suppressing the activity of various bacteria.

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