JP4919757B2 - Continuous production method of alcohol - Google Patents

Description

  The present invention relates to a method for continuously producing alcohol, and more particularly, to a method for continuously producing alcohol, in which the yield of ethanol production Yp / x per cell is much higher than that of conventional yeasts.

Conventionally, Patent Document 1 discloses a method for fermenting alcohol using yeast consisting of Saccharomyces cerevisiae AM12 strain (hereinafter referred to as AM12 strain if necessary). And has a strong cohesiveness.
JP 59-135896 A

  However, since alcoholic fermentation in Patent Document 1 is anaerobic culture (see Example 2 of the same document), it is not suitable for a continuous ethanol production process, and there is a disadvantage that ethanol productivity per unit time in the continuous fermentation process is insufficient. It was.

  Then, this invention makes it a subject to provide the continuous production method of alcohol with high continuous ethanol productivity under aerobic conditions.

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

  The above problems are solved by the following inventions.

(Claim 1)
Alcohol raw material is supplied to the calling酵槽performed alcohol fermentation, a continuous method of producing alcohol for continuous fermentation to alcohol withdrawal of the resulting alcohol culture from the fermentor,
Continuously supplying the alcohol raw material having a sugar concentration in the range of 100 to 300 g / L to the fermentor;
Saccharomyces cerevisiae (Saccharomyces cerevisiae) AM12 strain (Accession Number: FERM BP-798) as well as inoculation of yeast consisting in the fermenter at the start of the continuous fermentation, while maintaining the fermentation tank to be aerobically The yield Yp / x of ethanol per cell is represented by Yp / x = Yp / s / Yx / s (Yp / s is the ethanol yield, Yx / s cell yield). A continuous production method of alcohol , wherein the alcohol is continuously fermented in a range of x from 3.60 to 4.60 .

(Claim 2)
The continuous production method of alcohol according to claim 1, wherein the Yx / s is in the range of 0.10 to 0.20.

  ADVANTAGE OF THE INVENTION According to this invention, the continuous production method of alcohol with high continuous ethanol productivity can be provided under aerobic conditions.

  Embodiments of the present invention will be described below.

  First, the basic principle of the continuous alcohol production method according to the present invention will be described.

  The ethanol productivity per unit time in the continuous fermentation process is represented by the following formula (1).

Ethanol productivity (g / L / h): J = D · Yp / x · x (1)
D: Dilution rate (value obtained by dividing saccharified solution supply rate by culture volume)
Yp / x: ethanol production yield per cell
x: Cell density (g / L)

  Yp / x of conventional yeast (bakers yeast: literature value) is in the range of 0.84 (aerobic condition) to 3.54 (-) (anaerobic condition), but the yeast (AM12 strain, According to an example of a peculiar experiment (also referred to as the yeast of the present invention), Yp / x was 4.55 (−) (microaerobic condition) (see Example).

  Therefore, it has been found that the Yp / x of the yeast of the present invention is higher than the Yp / x of the conventional yeast, and thus has a high ethanol production capacity.

  The ethanol production yield Yp / x per microbial cell is represented by the following formula (2).

      Yp / x = Yp / s / Yx / s (Yp / s is the ethanol yield, Yx / s is the cell yield) (2)

  According to an example of an experiment obtained by the aerobic culture of the present inventors, Yp / s = 0.50 (−) and Yx / s = 0.146 (−) in the yeast of the present invention.

  On the other hand, conventional yeast (bakers yeast: literature values) is Yp / s = 0.3 to 0.4 (−), Yx / s = 0.14 (anaerobic) to 0.53 (aerobic). .

  In conventional aerobic culture of yeast, cell growth is more dominant than ethanol production, whereas in this yeast, the cell yield Yx / s under aerobic conditions is almost the same as that under conventional anaerobic conditions. Yes, it was found that the Yp / x value was much higher than that of the conventional yeast under anaerobic conditions.

  That is, in the following reaction formula (3) in aerobic culture, in the yeast of the present invention, ethanol production is dominant over cell growth, unlike the conventional yeast, and the yield of ethanol production per cell. It is estimated that this is because the Yp / x value is extremely high.

_{C 6 H 12 O 6 + N} 2-source + O 2 → cell + C 2 H 5 OH + CO 2 ( aerobic condition) (3)

  That is, the present inventors have found that the yeast of the present invention has a Yp / x value in a very high range even in aerobic culture, and the Yp / s and Yx / s in the above formula (2). From the relationship, it has been found that Yx / s is a value equivalent to that under the conventional anaerobic conditions of yeast, and by finding these findings, the present invention has been completed.

  Next, the constituent requirements of the continuous alcohol production method according to the present invention will be described below.

  The alcohol raw material used in the present invention is preferably a cereal-derived raw material. For example, rice, wheat, potato and the like have high sugar concentrations, and are preferred examples. The sugar concentration is preferably in the range of 100 to 300 g / L, more preferably in the range of 150 to 200 g / L, from the viewpoint of saccharification reaction efficiency of the grain raw material.

  The aerobic fermentation temperature is preferably 40 ° C. or less, more preferably in the range of 30 to 35 ° C., from the viewpoint of the optimum fermentation temperature for yeast. For temperature adjustment, adjustment means such as a heater and a cooler can be adopted, but there is no particular limitation.

  In this invention, when maintaining the inside of a fermenter on aerobic conditions, DO (dissolved oxygen concentration) in a fermenter has the preferable range of 0.1-3 ppm, More preferably, the range of 0.1-1 ppm It is. The DO can be adjusted by increasing / decreasing the air supply amount and the rotation speed of the stirring blade in the fermenter. For example, a technique of interlocking the DO meter and the air supply amount control means can be adopted.

  The pH is preferably in the range of 3 to 7, more preferably in the range of 3.5 to 5.0, from the viewpoint of the optimum pH of the yeast. As a pH adjustment method, a pH control system that adjusts pH by continuously supplying a pH adjusting agent based on data measured by a pH meter can be employed.

  The dilution rate D is preferably in the range of 0.1 to 0.25, more preferably in the range of 0.15 to 0.20. The dilution rate D is a value obtained by dividing the saccharified solution supply rate by the culture volume.

  The bacterial cell concentration x is preferably in the range of 20-40 g / L, more preferably in the range of 25-30 g / L. The bacterial cell concentration can be measured by absorbance measurement (turbidity measurement).

  The Saccharomyces cerevisiae AM12 strain used in the present invention has been deposited at the National Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology as No. 6749. For details, JP-A-59-135896 can be referred to.

  In the present invention, continuous fermentation refers to continuous removal of a product while continuously supplying a sugar solution to a fermenter, and a culture solution mixed with ethanol and yeast is continuously removed. Refers to things.

  In culture under aerobic conditions, the value of ethanol production yield Yp / x per cell is preferably in the range of 3.60 to 4.60, more preferably 3.61 to 3.63. It is a range, More preferably, it is 3.62.

  In the present invention, in the culture under aerobic conditions, Yx / s (bacterial cell yield) is preferably in the range of 0.10 to 0.20, more preferably 0.13 to 0.16. It is a range, More preferably, it is 0.14.

  Examples of the present invention will be described below, but the present invention is not limited to such examples.

Example 1
<1. Preparation of saccharified solution>
The saccharified solution used as a raw material for continuous production of ethanol is obtained by adding yeast medium components (vitamin and mineral sources, yeast extract, polypeptone) to a saccharified solution of about 20% in terms of monosaccharide (glucose) concentration (yeast extract, peptone). Is added at a ratio of 0.1 (w / w)%, 0.2 (w / w)% to the diluted solution), and further sterilized by autoclaving (120 ° C., 1 atm, 20 minutes). The thing which gave is used.

<2. AM12 yeast inoculum preparation method>
AM12 yeast inoculum is stored in a flask with baffle in a YPD medium (yeast extract 1%, peptone 2%, glucose 5%, medium pH 5.0) and frozen at -80 ° C in a sterilized flask medium. Inoculated (added frozen yeast suspension using a platinum loop) and cultured overnight at 30 ° C under aerobic conditions (shaking culture at a shaking speed of 160 rpm). The performed yeast culture solution was used.

<3. Continuous ethanol fermentation using AM12 yeast>
The experiment was conducted using the continuous ethanol fermenter shown in FIG.

  In FIG. 1, 1 is a sugar liquid supply tank and 2 is a fermenter. The sugar solution is supplied from the sugar solution supply tank 1 to the fermenter 2 using a supply pump 10.

  The fermenter 2 includes a pH controller 20, and when the measured value of the pH measuring device 21 is input and the pH is not within the range of the following control conditions, the pH adjusting pump 22 is operated, and 1N-HCl and 2N-NaOH is supplied from the tank 23 to the fermenter 2.

  Moreover, the fermenter 2 is equipped with the temperature controller 24, and when the measured temperature of the temperature measuring device 25 is input and the range of the following control conditions is exceeded, cooling water is supplied to the cooling pipe 26 provided in the outer periphery of the fermenter 2. The temperature can be adjusted by supplying and cooling, or heating with a heater.

  Furthermore, the fermenter 2 includes a DO controller 27 and a DO meter 28, and the dissolved oxygen (DO) concentration is controlled by increasing or decreasing the rotational speed of the aeration and the stirring blade 29 in the fermenter.

  In FIG. 1, 30 is an alcohol pump for taking out the produced alcohol.

  First, start up continuous ethanol fermentation. That is, the above 1. is added to the 3 L fermenter 2 shown in FIG. 1 L of the saccharified solution prepared in step 2 above is added. Inoculate the yeast inoculum created in step 1 into the fermenter from the inoculation port, and start ethanol fermentation with yeast.

  Table 1 shows the fermenter control conditions.

Next, about 40 hours after the start of ethanol fermentation, While adding the saccharified solution prepared in step ¹ to the fermentor at a rate of f = 0.1 [L / h] (dilution rate 0.1 [h ⁻¹ ]), the culture solution in the fermentor at the same rate at the same time Was taken out (start of continuous ethanol fermentation).

Continuous ethanol production was performed by increasing f at the start of the experiment and increasing it to a maximum of 0.18 [L / h] (dilution rate 0.18 [h ⁻¹ ]).

<4. Analytical method in continuous ethanol fermentation>
A culture solution in which ethanol and ethanol flowing out of a fermenter during continuous ethanol production were mixed was obtained. Cell growth of AM12 yeast is measured by using a spectrophotometer (shimadzu UV-mini 1240) to measure turbidity (absorbance of OD600), and then a conversion formula (cell concentration (g / L) from turbidity to cell concentration. ) = 0.38 × OD600).

  The consumption situation of glucose accompanying ethanol fermentation was measured by using a glucose kid (Wako Pure Chemical Co., Ltd., Glucol Test-c-Wako).

  The time course of ethanol production was measured by a gas chromatograph (shimadzu GC-2014) equipped with an FID detector.

<5. Experimental results in continuous ethanol fermentation>
Table 2 shows the cell yield, ethanol yield, and ethanol production yield when continuous ethanol fermentation was performed using the AM12 strain by the above method.

By this continuous ethanol production method, the maximum ethanol productivity was 18.3 g / L at f = 0.18 [L / h] (dilution rate 0.18 [h ⁻¹ ]).

Comparative Example 1
Table 3 shows the cell yield, ethanol yield, and ethanol production yield when continuous ethanol fermentation was carried out using normal yeast (Saccharomyces cerevisiae JCM7255 standard yeast strain) in the same manner as in Example 1.

By this continuous ethanol production method, the maximum ethanol productivity was 13.3 g / L at f = 0.18 [L / h] (dilution rate 0.18 [h ⁻¹ ]).

Schematic diagram of continuous ethanol fermenter

Explanation of symbols

1: Sugar solution supply tank 10: Supply pump 2: Fermenter 20: pH controller 21: pH measuring device 22: pH adjusting pump 23: Tank 24: Temperature controller 25: Temperature measuring device 26: Cooling pipe 27: DO controller 28: DO meter 29: stirring blade 30: alcohol pump

Claims (2)

Alcohol raw material is supplied to the calling酵槽performed alcohol fermentation, a continuous method of producing alcohol for continuous fermentation to alcohol withdrawal of the resulting alcohol culture from the fermentor,
Continuously supplying the alcohol raw material having a sugar concentration in the range of 100 to 300 g / L to the fermentor;
Saccharomyces cerevisiae (Saccharomyces cerevisiae) AM12 strain (Accession Number: FERM BP-798) as well as inoculation of yeast consisting in the fermenter at the start of the continuous fermentation, while maintaining the fermentation tank to be aerobically The yield Yp / x of ethanol per cell is represented by Yp / x = Yp / s / Yx / s (Yp / s is the ethanol yield, Yx / s cell yield). A continuous production method of alcohol , wherein the alcohol is continuously fermented in a range of x from 3.60 to 4.60 . The continuous production method of alcohol according to claim 1, wherein the Yx / s is in the range of 0.10 to 0.20.

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