JP4854568B2 - Alcohol production method - Google Patents

Description

  The present invention relates to an alcohol production method, and more particularly to an alcohol production method that can be quickly recovered even when contaminated with various bacteria during continuous production of alcohol for recycling bacterial yeast.

  The continuous ethanol production method that produces ethanol continuously has high ethanol productivity, but ethanol production efficiency fluctuates greatly depending on environmental factors such as ethanol tolerance of yeast and oxygen conditions, so stable and highly efficient ethanol production is possible. It was difficult.

  In particular, the alcohol raw material is continuously fed into the fermenter to perform alcoholic fermentation, the obtained alcohol solution is extracted from the fermenter, and the yeast cells in the extracted alcohol solution are precipitated in a settling tank. The alcohol continuous production method in which the cells are recycled, separated and recovered and returned to the fermenter, has a high possibility of mixing microorganisms other than yeast (miscellaneous bacteria) because the production process becomes complicated.

  In alcohol production, ethanol, which is a product, has an inhibitory effect on living organisms, so even if microorganisms that are not resistant to alcohol are mixed, they cannot be proliferated in an alcoholic solution and are therefore hardly contaminated. However, some bacteria belonging to lactic acid bacteria, commonly called “burning fungi”, are generally resistant to 15% ethanol and can grow, and some can also grow in 25% ethanol. It is known as a major pollutant in (Non-patent Document 1).

Conventionally, when contaminated with various bacteria, stop production, discard all alcohol and yeast cells, wash the equipment, sterilize and re-install, inoculate with new yeast inoculum and start up again Is common. If a long period of time is required until the original state is restored, industrial production is reduced accordingly.
Tamura, Kazuo, Nohaku, Kikuo, Akiyama, Yuichi and Koizumi, Takeo, “Challenge from Yeast: Applied Yeastology”, Gihodo Publishing, 1997, p. 87-90

  In the alcohol production in which bacterial cells are recycled, the present inventors consider that the loss due to contamination of various bacteria can be reduced if there is a method for recovering from the state contaminated with various bacteria in a short period of time, and intensive research has been conducted. As a result, the present invention has been reached.

  Accordingly, an object of the present invention is to provide a method for producing alcohol that, when contaminated with various germs, quickly removes the germs and recovers from the contaminated state in a short period of time.

  The other subject of this invention becomes clear by the following description.

  The above problems are solved by the following invention.

(Claim 1)
Using yeast consisting of Saccharomyces cerevisiae AM12 strain, the alcohol raw material is continuously fed into the fermenter to perform alcoholic fermentation, and the resulting alcohol solution is withdrawn from the fermenter and then withdrawn alcohol. In the alcohol production method in which the cells of AM12 bacteria in the liquid are precipitated in a sedimentation separation tank, separated and recovered, and returned to the fermentation tank, in which alcohol is recycled.
The supernatant liquid separated in the settling tank is removed, and the washing liquid having an ethanol concentration of 14% to 20% and pH of 6 to 8 is added to and mixed with the AM12 bacteria remaining after precipitation, and then for a certain period of time. A method for producing alcohol, characterized by recovering the contamination of AM12 bacteria by allowing to stand to precipitate the cells of AM12 bacteria and removing the supernatant.

(Claim 2)
When the washing liquid is added to and mixed with the cells of AM12, the return of the cells of AM12 from the sedimentation tank to the fermentation tank and the feeding of the alcohol liquid from the fermentation tank to the sedimentation tank are stopped. The alcohol production method according to claim 1, wherein:

(Claim 3)
3. When the washing liquid is added to and mixed with the cells of AM12 in the sedimentation separation tank, it is allowed to stand for 5 minutes to 40 minutes, and then the supernatant liquid is extracted from the sedimentation separation tank. The alcohol production method as described.

(Claim 4)
The alcohol production method according to any one of claims 1 to 3, wherein the washing for eliminating germs is repeated one or more times according to the degree of contamination.

  ADVANTAGE OF THE INVENTION According to this invention, when contaminated with various germs, the alcohol production method which removes germs rapidly and recovers from a contaminated state in a short period of time can be provided.

  Embodiments of the present invention will be described below.

  The alcohol production method of the present invention uses a yeast having a coagulation sedimentation property, preferably a yeast consisting of Saccharomyces cerevisiae AM12 strain (hereinafter referred to as AM12 strain), continuously in a fermenter. The obtained alcohol solution is extracted from the fermenter, and the yeast cells in the extracted alcohol solution are precipitated and separated and collected in a settling tank, and returned to the fermenter. This is a method for recovering from a contaminated state in a short time when contaminated with various bacteria in a continuous alcohol production method in which cell bodies are recycled.

  In the present invention, the alcohol production is to continuously take out the product while continuously supplying the sugar solution to the fermenter, and to continuously take out the culture solution mixed with ethanol and yeast. Point to.

  In the present invention, “recycling bacterial cells” means that the culture solution mixed with ethanol and yeast taken out from the fermenter is allowed to stand to precipitate and separate the yeast cells, regardless of whether or not re-cultured. It is defined as performing alcoholic fermentation by returning a part of the separated cells to the fermenter.

  The alcohol production system includes a sugar solution supply tank, a fermentation tank, a sedimentation separation tank, and lines connecting them, and has a structure in which microorganisms are not easily mixed from the outside.

  In the present invention, “miscellaneous bacteria” refers to microorganisms other than yeast that are mixed in the alcohol production system for some reason, and “contaminated by miscellaneous bacteria” means microorganisms other than yeast in the alcohol solution. Is a state that is proliferated and detected, and “recovering the contamination of germs” means that microorganisms other than yeast are removed from the alcohol production system so that continuous alcohol production is performed in the same manner as before the contamination. It is to be.

  The agglutinating sedimentary yeast AM12 used in the present invention has been deposited with the Industrial Technology Microbiology Industrial Technology Research Institute as a fine work laboratory accession number 6749. For details, JP-A-59-135896 can be referred to.

  The alcohol production method of the present invention will be described based on FIG.

  In FIG. 1, 1 is a sugar liquid supply tank, 2 is a fermenter, and 3 is a sedimentation / separation tank for separating cells from the alcohol liquid.

  The raw material is supplied from the sugar solution supply tank 1 to the fermenter 2 through a supply pump 11 and a supply line 12. From the supply line 12, in addition to the sugar solution as a raw material, a nutrient solution necessary for cell growth such as peptone or a trace metal salt solution can be added.

  The fermenter 2 includes a stirrer 21 for sufficiently mixing the cells and the supplied raw material. The stirring device may be any device as long as the mixing in the fermenter is sufficiently performed, but a stirring device having a stirring blade such as a propeller is preferable, and the rotation speed of stirring is adjusted to be an optimum value for yeast. Moreover, although not shown in figure, a fermenter can be equipped with the system which is not specifically limited in order to maintain pH, temperature, and dissolved oxygen concentration.

  In the fermenter 2, alcoholic fermentation is performed by yeast, and the raw sugar solution becomes a liquid (culture solution) containing alcohol and yeast cells, and is sent to the sedimentation / separation tank 3 from the collection pump 22 and the collection line 23. In addition, the gas generated along with the alcohol fermentation is discharged from the line 24.

  In the sedimentation / separation tank 3, sedimentation / separation is performed by standing, and the coagulating sedimentary yeast settles and is separated from the alcohol liquid. In FIG. 1, 3a is a cell body of precipitated AM12 bacteria (yeast), 3b is a supernatant liquid from which yeast cells have been removed, and refers to an alcohol liquid when normal alcohol production is performed. The alcohol liquid 3b is extracted by the alcohol pump 31 and sent to the next purification process through the alcohol line 32.

  A part of the settled and concentrated cells 3a is taken out, and a specified amount of cells are returned to the fermenter 1 from the supply line 12 together with the raw materials through the recycle pump 33 and the recycle line 34 connected to the supply line 12. Further, excess cells (precipitate) are discharged from the discharge line 35.

  In FIG. 1, reference numeral 4 denotes a cleaning liquid tank for containing a cleaning liquid for cleaning the sedimentation / separation tank 3 when contaminated with bacteria, 41 is a cleaning liquid supply pump, and 42 is a cleaning liquid supply line.

  In FIG. 1, the cleaning liquid feeding line 42 is connected to the lower part of the sedimentation separation tank 3, but the cleaning liquid is connected to the recycling line 34 and the washing liquid is supplied to the sedimentation separation tank 3 so as to flow backward from the recycling line 34 to the normal flow path. It can also be injected. A control unit 5 controls the alcohol continuous production system pump.

  Below, the removal method of the germ which recovers the germ contamination in this invention is demonstrated.

  In order to monitor contamination due to various germs, the culture solution sent from the collection line 23 to the sedimentation tank 3 is periodically sampled and inspected.

  Since a rapid judgment is required for the test, a molecular biological test method is preferable to a test method such as culture. Specifically, DNA is extracted and amplified using PCR, and the amplified DNA is amplified. For example, a method of detecting a gene by electrophoresis of fragments and detecting the presence or absence of a band can be used, and commercially available test kits can be used for major contaminants such as fire-fung bacteria.

  The detection of miscellaneous bacteria is determined by whether or not the gene of miscellaneous bacteria is detected from the sampled alcohol solution.

  When the gene is detected and it is found that there is contamination by various bacteria, the control unit 5 stops the supply pump 11, the liquid collection pump 22, and the recycle pump 33 to supply the sugar solution to the fermenter and the fermenter 2. The supply of the culture solution in the sedimentation / separation tank 3 and the supply of yeast cells from the sedimentation / separation tank 3 to the fermenter 2 are stopped.

  When the inflow of the liquid in the sedimentation / separation tank 3 stops, the AM12 bacteria settle quickly because of high coagulation sedimentation. Since the contaminated microorganism does not settle as quickly as AM12 bacteria, it exists in the supernatant 3b. In this state, the control unit 5 operates the alcohol pump 31 to extract the supernatant 3b from the sedimentation tank 3 and discard it.

  That is, AM12 bacteria can be separated from miscellaneous bacteria having different sedimentation rates without using a centrifuge by utilizing the strong aggregation sedimentation property of AM12 bacteria.

  For this reason, after the inflow of liquid into the sedimentation / separation tank 3 is stopped, the time for sedimentation of AM12 bacteria is 5 minutes to 40 minutes, preferably 10 minutes to 30 minutes, and then promptly. It is necessary to draw out the supernatant 3b.

  After wiping off and discarding the alcohol culture solution of the supernatant, the control unit 5 operates the washing solution feed pump 41 to inject the washing solution in the washing solution tank 4 from the lower part of the sedimentation separation tank 3.

  The composition of the cleaning solution is adjusted to an ethanol concentration of 14% to 20%, preferably 14.5% to 17%, a pH of 6 to 8, preferably 6.5 to 7.5, and sterilized with an autoclave or a filter. Is preferred.

  AM12 bacteria have ethanol resistance that is not inhibited even at an ethanol concentration of 20%, but most other microorganisms cannot survive or grow in a 20% ethanol liquid.

  Among Lactobacillus, Lactobacillus homohichii, which has particularly high ethanol resistance, Bacteria said to be a true fire bacterium such as Frutivorans have a low growth inhibitory effect at the preferred ethanol concentration of the washing solution of the present invention, but these bacteria have an optimum pH of growth of 4.5 to 5.0, and at pH 5.5 or higher. Since it cannot grow, the cleaning solution of the present invention having an ethanol concentration of 14% to 20%, preferably 14.5% to 17%, pH of 6 to 8, preferably 6.5 to 7.5 is Even if it is effective.

  The cleaning liquid pump 41 floats the AM12 bacterial cells 3a that settle in the sedimentation separation tank 3 at a flow rate that allows the AM12 bacterial cells to sufficiently contact and stir the cleaning liquid in the sedimentation separation tank 3. It is preferable to have an injectable output, and a cleaning solution of about 50 to 80% of the sedimentation tank capacity is injected in one cleaning. This is because the effect of washing various bacteria is enhanced by floating the precipitated yeast cells.

  When the washing liquid injection is completed and the stirring is stopped, the AM12 bacteria quickly settle. Similarly, the AM12 bacteria are allowed to settle for 5 minutes to 40 minutes, preferably 10 minutes to 30 minutes, and then quickly The pump 31 is operated and the supernatant 3b is extracted.

  A sample of the extracted supernatant 3b is sampled and examined for detection of germ genes. If no germ gene is detected, normal alcohol continuous production is started on the assumption that the removal of the germ has been completed (recovered from contamination). If detected, the washing solution is injected again and the washing process is performed until no bacteria are detected.

  FIG. 2 shows a control process performed by the control unit 5 for the above-described cleaning process.

  The sampled culture solution is inspected and inspection data is input (S1). It is determined from the inspection data (for example, whether or not a band is detected) whether there is any contamination (S2). If it is determined that there is no contamination, the process ends.

  If it is determined that there is contamination, the supply pump 11, the intake pump 22 and the recycle pump 33 are stopped to precipitate AM12 bacteria, and then the alcohol pump 31 is operated to obtain supernatant from the sedimentation separation tank 3. The alcohol liquid 3b is extracted (S3).

  After extracting the alcohol liquid, the cleaning liquid feed pump 41 is operated, the cleaning liquid in the cleaning liquid tank 4 is injected from the lower part of the sedimentation separation tank 3, and the cells 3a of the AM12 bacteria that precipitate are suspended (S4).

  After the AM12 bacteria are precipitated, the alcohol pump 31 is operated and the supernatant washing liquid 3b is extracted from the sedimentation tank 3 (S5).

  The cleaning liquid 3b is sampled and inspected, and inspection data is input (S6). It is determined from the inspection data (for example, whether or not a band is detected) whether or not there are any germs (S7). If miscellaneous bacteria exist, the process returns to S4.

  When the presence of miscellaneous bacteria is not recognized, supply pump 11, take-up pump 22, recycle pump 33, and alcohol pump 31 are sequentially operated to resume the supply of sugar solution to the fermenter and normal continuous alcohol production. (S8).

  In the method of the present invention, it is possible to remove miscellaneous bacteria with almost no loss of yeast cells. Therefore, in alcohol production, it is possible to shorten the period from elimination of miscellaneous bacteria to normal operation.

  EXAMPLES Hereinafter, although this invention is demonstrated based on an Example, this invention is not limited by this Example.

  A medium containing 20% by weight of monosaccharide (converted to glucose) and 0.1% by weight of corn steep liquor was placed in a heat-sterilized fermenter, inoculated with an inoculum of AM12, and ethanol fermentation was started.

About 40 hours after the start of ethanol fermentation, while continuously supplying the sugar solution to the fermenter, continuous ethanol production was started to continuously take out the product, and the dilution rate was finally set to 0.18 h ^−1. It was confirmed that the concentration of each component of yeast cells, ethanol, and glucose in the fermenter was in a constant state (steady state), and continuous alcohol production was started.

  In order to create a state where the alcohol production system was intentionally contaminated with various germs, fire-fung bacteria (L. homohichi RIB-9134 RIKEN BioResource Center No. JCM7524) were mixed in the fermentor and continuous production was performed for 72 hours.

  After 72 hours, the alcohol culture solution was sampled from the fermentor, centrifuged to collect the cells, and then the DNA was extracted and examined using the Takara PCR Fire Detection Kit (Takara Bio Inc.). A dark band was confirmed, and fire eradication bacteria were detected.

  With respect to the alcohol production system contaminated with this germ, an attempt was made to recover from the contamination by using the present invention.

  Since miscellaneous bacteria were detected, the supply pump 11, the intake pump 22 and the recycle pump 33 were stopped and allowed to stand for 30 minutes to precipitate AM12 bacteria.

  Thereafter, the alcohol pump 31 was operated, and the supernatant alcohol liquid 3b was quickly extracted from the sedimentation tank 3.

  After extracting the alcohol liquid, the cleaning liquid feed pump 41 was operated, and the cleaning liquid in the cleaning liquid tank 4 was injected from the lower part of the settling separation tank 3 to float the AM12 bacterial cells 3a to be precipitated.

  The cleaning solution was adjusted to pH 7 and ethanol concentration 15%.

  After allowing to stand for 30 minutes to precipitate AM12 bacteria, the alcohol pump 31 was operated, and the supernatant washing liquid 3b was extracted from the sedimentation tank 3.

  When the cleaning solution was sampled and tested for fire bacteria, a thin band was confirmed and a small amount of fire bacteria was detected. The amount of fire-fung bacteria detected was reduced by washing.

  The cleaning liquid was again injected and cleaning operation was performed. When the cleaning solution was sampled and tested for fire-fung bacteria, no band was confirmed. Therefore, it was possible to remove the fire bacteria.

  Since the presence of various germs was not recognized, normal supply continuous production was started by sequentially operating the supply pump 11, the liquid collecting pump 22, the recycle pump 33, and the alcohol pump 31.

Schematic of continuous alcohol production of the present invention The figure which shows the control processing which a control part performs

Explanation of symbols

1: Sugar solution supply tank 11: Supply pump 12: Supply line 2: Fermenter 21: Stirrer 22: Extraction pump 23: Extraction line 24: Line 3: Sedimentation separation tank 3a: Cell body of AM12 bacteria 3b: Supernatant Liquid (alcohol liquid, cleaning liquid)
31: Alcohol pump 32: Alcohol line 33: Recycle pump 34: Recycle line 35: Discharge line 4: Cleaning liquid tank 41: Cleaning liquid feeding pump 42: Cleaning liquid feeding line 5: Control unit

Claims (4)

Using yeast consisting of Saccharomyces cerevisiae AM12 strain, the alcohol raw material is continuously fed into the fermenter to perform alcoholic fermentation, and the resulting alcohol solution is withdrawn from the fermenter and then withdrawn alcohol. In the alcohol production method in which the cells of AM12 bacteria in the liquid are precipitated in a sedimentation separation tank, separated and recovered, and returned to the fermentation tank, in which alcohol is recycled.
The supernatant liquid separated in the settling tank is removed, and the washing liquid having an ethanol concentration of 14% to 20% and pH of 6 to 8 is added to and mixed with the AM12 bacteria remaining after precipitation, and then for a certain period of time. A method for producing alcohol, characterized by recovering the contamination of AM12 bacteria by allowing to stand to precipitate the cells of AM12 bacteria and removing the supernatant.   When the washing liquid is added to and mixed with the cells of AM12, the return of the cells of AM12 from the sedimentation tank to the fermentation tank and the feeding of the alcohol liquid from the fermentation tank to the sedimentation tank are stopped. The alcohol production method according to claim 1, wherein:   3. When the washing liquid is added to and mixed with the cells of AM12 in the sedimentation separation tank, it is allowed to stand for 5 minutes to 40 minutes, and then the supernatant liquid is extracted from the sedimentation separation tank. The alcohol production method as described.   The alcohol production method according to any one of claims 1 to 3, wherein the washing for eliminating germs is repeated one or more times according to the degree of contamination.

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