JP5700564B2 - Ethanol-fermenting yeast with excellent salt resistance, heat resistance, ethanol resistance, and coagulation sedimentation, and use thereof - Google Patents

Description

  The present invention relates to an ethanol fermentable yeast excellent in salt resistance, heat resistance, ethanol resistance, and coagulation sedimentation, and a method for producing ethanol using the yeast.

  In the fermentation production of ethanol, in order to lower the production cost, it is important to provide inexpensive fermentation raw materials, improvement of fermentation yield, reduction of cooling water, reduction of distillation energy, and the like (Non-Patent Documents 1 and 2). Among these, improvement of fermentation efficiency, reduction of cooling water, and reduction of distillation energy largely depend on the characteristics of yeast used for fermentation. About improvement of fermentation efficiency so far, high ethanol productivity, heat resistance, acid resistance (Patent Documents 1 and 4), sugar resistance, ethanol resistance (Patent Document 2), salt resistance (Patent Document 3), coagulation sedimentation Yeast has been developed from the viewpoints of Patent Documents 4 and 5. Moreover, about the reduction | decrease of cooling water and the reduction | decrease of distillation energy, development of the yeast (patent documents 4 and 5) which can be fermented on high temperature conditions is known.

  Waste molasses are known as an inexpensive raw material suitably used for ethanol fermentation. Waste molasses is a viscous dark brown liquid after squeezing crude sugar from raw sugar such as sugarcane. Waste molasses contains various salts such as potassium, calcium, and magnesium, and the amount thereof varies depending on the raw material production area and the number of times the raw sugar is squeezed, but the concentration is generally 1.5 to 4.5M. For this reason, in the fermentation production of ethanol using molasses as a raw material, it is essential to have salt tolerance, and furthermore, the above-mentioned high ethanol productivity, heat resistance, acid resistance, sugar resistance, ethanol resistance, and coagulation sedimentation properties. Development of yeast having such is desired.

JP 2009-171912 A Japanese Patent Laid-Open No. 10-262651 Japanese Patent Publication No. 7-32701 JP-A-5-236842 JP 59-135896 A

Illustrated Bioethanol Production Technology Alcohol Association edited by Industrial Research Committee Co., Ltd. (2007) Illustrated front line of bioethanol Yasuhiro Daisei, edited by Mitsui & Co., Ltd., Industrial Research Committee, Inc. (2004)

  An object of the present invention is to provide a yeast having high salt tolerance and further having high ethanol productivity, heat resistance, ethanol resistance, and coagulation sedimentation, and an ethanol production method using the yeast.

The present invention includes the following inventions in order to solve the above problems.
[1] (a) Can be grown on YPD medium containing 2.0 M KCl, (b) Diluted molasses containing 15% (w / w) sugar and 5-6% (w / w) ash Using the liquid as a raw material, 7-10% (v / v) ethanol was produced at a culture temperature of 40 ° C. (C) After stirring and culturing at 35 ° C for 12 hours in a YPD medium containing 5% glucose, stirring was stopped. A yeast that aggregates and settles within 1 minute from the start.
[2] Using a waste molasses diluted solution containing 15% (w / w) sugar and 5-6% (w / w) ash as raw materials, repeated batch culture of 8 or more times at a culture temperature of 38 ° C. is possible. Yeast as described in said [1] characterized by these.
[3] Using a waste molasses diluted solution containing 15% (w / w) sugar and 5-6% (w / w) ash as a raw material, it is possible to cultivate four or more times at a culture temperature of 40 ° C. The yeast according to [1] or [2] above, wherein
[4] The yeast according to any one of [1] to [3] above, which belongs to Saccharomyces cerevisiae.
[5] The yeast according to [4] above, which is Saccharomyces cerevisiae BA11 strain (Accession No. NITE BP-793) or Saccharomyces cerevisiae BA16 strain (Accession No. NITE BP-794).
[6] A method for producing ethanol, comprising using the yeast according to any one of [1] to [5].
[7] The method for producing ethanol according to [6] above, wherein a raw material containing molasses is used.

  According to the present invention, it is possible to provide a yeast having high salt resistance, high ethanol productivity, heat resistance, ethanol resistance, and coagulation sedimentation, and an ethanol production method using the yeast. If the yeast of the present invention is used, the fermentation yield can be improved, the cooling water can be reduced and the distillation energy can be reduced, and further the yeast centrifugation step can be omitted, thus greatly reducing the production cost of bioethanol. It becomes possible. Moreover, since ethanol can be efficiently produced using waste molasses or the like having a high salt content as a raw material, the raw material cost can be greatly reduced.

It is a figure which shows the result of having examined the growth of the various yeast in the YPD agar medium containing 2.0M KCl. It is a figure which shows the result of having examined the growth of the various yeast in the YPD agar medium which does not contain KCl. It is a figure which shows the result of having examined the aggregation sedimentation property of BA11 stock | strain. It is a figure which shows the result of having investigated the aggregation sedimentation property of BA11 stock | strain and BA16 stock | strain. It is a figure which shows the result of having compared the fermentation performance of BA11 stock | strain and the conventional stock | strain A. FIG. It is a figure which shows the result of having examined the repeated fermentation performance in 40 degreeC of BA11 stock | strain. It is a figure which shows the result of having examined the fermentation performance of BA11 stock | strain and BA16 stock | strain. It is a figure which shows the result of having examined the heat resistance of BA11 stock | strain. It is a figure which shows the result of having examined the heat resistance of BA11 stock | strain and BA16 stock | strain, (A) is a figure which shows the result which confirmed the growth in 38 degreeC, (B) is 43 degreeC. It is a figure which shows the result of having analyzed the polyploidy of BA11 stock | strain and BA16 stock | strain. (A) is a figure which shows the rDNA area | region of yeast, (B) is a figure which shows the result of having analyzed the base sequence of the rDNA area | region of BA11 strain | stump | stock and BA16 strain | stump | stock.

〔yeast〕
The yeast of this invention should just have the following characteristics (a), (b), (c).
(A) It can grow on a YPD medium containing 2.0 M KCl.
(B) Using a waste molasses diluted solution containing 15% (w / w) sugar and 5-6% (w / w) ash as a raw material, 7-10% (v / v) ethanol at a culture temperature of 40 ° C. Produce.
(C) After stirring and culturing at 35 ° C. for 12 hours in a 5% glucose-containing YPD medium, the mixture is agglomerated and settled within 1 minute after the stirring is stopped.

The yeast of the present invention preferably further has the following characteristics (d) and (e).
(D) The waste molasses dilution containing 15% (w / w) sugar and 5-6% (w / w) ash can be used as a raw material for repeated batch cultures of 8 or more times at a culture temperature of 38 ° C.
(E) Using a waste molasses diluted solution containing 15% (w / w) sugar and 5-6% (w / w) ash as raw materials, it is possible to perform four or more repeated batch cultures at a culture temperature of 40 ° C.

  The above-mentioned property (a) can be confirmed, for example, by inoculating yeast on a YPD agar medium containing 2.0 M KCl and culturing at about 35 ° C. for about 24 hours. Specifically, YPD agar medium supplemented with KCl to 2.0 M (yeast extract 1% (w / v), peptone 2% (w / v), glucose 2% (w / v), agar 1 0.5-2% (w / v)), inoculated with yeast and allowed to stand for about 24 hours in an incubator set at about 35 ° C., and then the presence or absence of yeast growth may be confirmed. .

  Having the characteristics of (b) above is that yeast is added to a waste molasses diluted solution containing 15% (w / w) sugar and 5-6% (w / w) ash, and cultured at a culture temperature of 40 ° C. This can be confirmed by measuring the ethanol concentration in the culture solution. Here, the molasses is a viscous black-brown liquid obtained by squeezing crude sugar from raw sugar such as sugarcane, and the molasses dilution can be prepared by diluting the molasses with water. Sugar and ash can be measured by known methods. Examples of the method for measuring sugar content include a high performance liquid chromatography method, and examples of the method for measuring ash content include a method in which waste molasses is ashed by heating at about 600 ° C. and the weight thereof is measured. . The ethanol concentration can be measured by a known method such as a gas chromatography method.

The culture conditions other than the culture temperature being 40 ° C. are not limited. Although an example of the culture method is shown below, it is not limited.
(1) Pre-culture; using a 500 ml baffled Erlenmeyer flask, two platinum ear plantings of a cryopreserved yeast strain (for example, a yeast strain stored at −80 ° C. in 15% glycerol) in 100 mL of YPD medium containing 5% glucose Inoculate and incubate for 24 hours at 38 ° C. and 170 rpm. Subsequently, the entire amount of the culture solution is put into a 5 L jar fermenter containing 3 L of 5% glucose-containing YPD medium, and cultured for 24 hours under conditions of 38 ° C., 200 rpm stirring, and 0.5 VVM aeration.
(2) Main culture: Put 100L of waste molasses diluted with water so that the sugar content becomes 10% (w / w) (100% final concentration added with ammonium sulfate) into a 100L liquor tank. 3 L of the preculture solution is added to the culture medium, and cultured for 24 hours under conditions of 38 ° C., 20 HZ stirring, and 0.5 VVM aeration.
(3) Fermentation: Waste molasses diluted with water to a 10,000L fermenter with a sugar content of 15% (w / w) and an ash content of 5-6% (w / w) (addition of ammonium sulfate at a final concentration of 0.8%) 4275 L is added, and 214 L of the above cultured bacterial cell solution is put therein, and cultured for 17 hours under conditions of 40 ° C., gentle stirring (150 rpm), and aeration / circulation rate of 15 m ³ / hr.

  It can confirm that it has the characteristic of said (c) by inoculating yeast to YPD culture medium containing 5% of glucose, and stopping stirring after performing stirring culture at 35 degreeC for 12 hours. Specifically, 100 mL of 5% glucose-containing YPD medium (yeast extract 1% (w / v), peptone 2% (w / v), glucose 5% (w / v)) is placed in a 200 mL flask, and yeast Is inoculated (about 0.5 platinum-shaped ears), and stirred culture is performed at 35 ° C. and 150 rpm, and stirring is stopped after 12 hours from the start of the culture. The culture may be transferred to a suitable container such as a beaker or a graduated cylinder and allowed to stand, and after 1 minute, it may be confirmed that the bacterial cell layer that has agglomerated and sedimented and the liquid layer without turbidity are separated.

  Having the above-mentioned characteristic (d) is that yeast is added to a dilute molasses solution containing 15% (w / w) sugar content and 5-6% (w / w) ash content, and batch culture is performed at a culture temperature of 38 ° C. This can be confirmed by repeating this eight times or more. A culture method in which a new medium is prepared for each culture and the medium is not added until the end of the culture is called batch culture. Repeated batch culture uses yeast coagulation sedimentation, separates yeast and supernatant without centrifuging after culturing, and adds a new medium to the separated yeast for the next culture. Is a culture method. “8 or more repeated batch cultures are possible” means that ethanol productivity can be maintained even when 8 or more repeated batch cultures are performed. “The state in which ethanol productivity does not decrease” is based on the fact that the ethanol production at that time is 70% or more of the highest ethanol production before the previous time. Specifically, a waste molasses diluted solution containing prescribed sugar and ash is prepared by diluting the molasses with water, and yeast is added using a suitable culture vessel and cultured at 38 ° C. After completion of the culture, the yeast is coagulated and settled to collect the supernatant (moromi), and a freshly diluted molasses solution is added to the separated yeast to start the second culture. This is repeated 8 times or more, and the ethanol concentration in the supernatant collected each time is measured to confirm that the state where ethanol productivity is not lowered is maintained.

  Having the above characteristic (e) is that yeast is added to a dilute molasses solution containing 15% (w / w) sugar and 5-6% (w / w) ash to batch culture at a culture temperature of 40 ° C. This can be confirmed by repeating this process four or more times. Specifically, in the method for confirming the characteristics of (d) above, the culture temperature may be changed to 40 ° C. and the number of repetitions may be changed to 4 times or more.

  Examples of the yeast of the present invention include, for example, Saccharomyces, Candida, Torulopsis, Zygosaccharomyces, Schizosaccharomyces, Pichia, Yarrowia, Hansenula, Kluyveromyces, Debaryomyces, Geotrichum, And those belonging to the genus Sporobolomyces, and those belonging to the genus Saccharomyces are preferred, and Saccharomyces cerevisiae is particularly preferred.

  The yeast of the present invention is screened for natural yeast or yeast produced by a known breeding method (for example, a mutagenesis method, a cell fusion method, a sexual conjugation method, etc.), and the above (a), (b) and It can be obtained by selecting one having the characteristic (c). Examples of such yeast include Saccharomyces cerevisiae BA11 strain produced by the inventor by a mutation induction method and Saccharomyces cerevisiae BA16 strain produced by a cell fusion method. Saccharomyces cerevisiae BA11 strain (Accession No. NITE BP-793) and Saccharomyces cerevisiae BA16 strain (Accession No. NITE BP-794) are respectively incorporated by the National Institute of Technology and Evaluation Technology Microorganisms (Chiba, Japan 292-0818) It has been deposited internationally in Kisarazu City, Kazusa Kamashiga 2-5-8) (date of trust: August 11, 2009).

[Ethanol production method]
The ethanol production method of the present invention may be any method that uses the yeast of the present invention. Therefore, individual specific steps in ethanol production are not particularly limited. For example, it can select suitably according to the raw material etc. which are used from the manufacturing method of the well-known bioethanol described in the nonpatent literature 1, the nonpatent literature 2, etc., and can use.

  Since the yeast used in the ethanol production method of the present invention has high ethanol productivity, ethanol can be efficiently produced using any raw material. For example, sugar raw materials such as sugarcane, sugar beet, and sugarcane-derived waste molasses; starchy raw materials such as corn, potato, sweet potato, rice, and wheat; cellulosic biomass can be preferably used. It is not something. Since the yeast used for the ethanol production method of the present invention has high salt tolerance, a raw material having a high salt concentration can be suitably used. Examples of the raw material having a high salt concentration include sugarcane-derived molasses. The salt concentration in sugarcane-derived molasses varies depending on the place of production, the state of recovery of crude sugar, etc. Magnesium ion concentration 0.7-1.1% (w / w), potassium ion concentration 5.6-9.4% (w / w), calcium concentration 1.1-1.8% (w / w) w), which contained about 15-20% (w / w) as the ash concentration. According to the ethanol production method of the present invention, it is possible to use sugarcane-derived waste molasses originally of low utility value for ethanol fermentation, so that raw material costs can be reduced.

  Since the yeast used for the ethanol production method of the present invention has excellent heat resistance, the fermentation process can be performed at a high temperature of about 38 to 40 ° C. Since the conventional strain has low heat resistance, it is necessary to perform the fermentation process at 30 to 33 ° C. Therefore, it has been essential to cool the fermenter using a cooling facility (chiller). According to the ethanol production method of the present invention, it is sufficient to cool with groundwater or the like even in the summer, so that it is possible to greatly save the power cost for cooling. The reduction in cooling costs is particularly noticeable for ethanol production in tropical subtropical areas. Furthermore, when the cellulose raw material is saccharified with cellulase, the higher the temperature, the higher the activity of cellulase. Therefore, the yeast of the present invention can be suitably used in an ethanol production method in which saccharification and ethanol fermentation are simultaneously performed.

  Since the yeast used in the ethanol production method of the present invention has excellent ethanol resistance and coagulation sedimentation properties, the repeated batch culture method (also referred to as “repetitive batch fermentation method”) is suitably employed in the ethanol production method of the present invention. can do. By adopting the repeated batch culture method, the step of centrifuging yeast can be omitted, and the next fermentation cycle can be started immediately by simply removing the mash and adding the raw material. Furthermore, since it is not necessary to make a liquor every time, it is possible to greatly reduce the manufacturing cost.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to these.

[Example 1: Production of Saccharomyces cerevisiae BA11 strain]
Saccharomyces cerevisiae BA6 strain with excellent alcohol resistance, high temperature resistance, and coagulation sedimentation among the strains stored in Bio Academia Co., Ltd., known mutation induction method (Yasuharu Oshima, Molecular genetics experiment method of yeast, Academic Publishing Center) In order to select a strain having high salt resistance and heat resistance after adding EMS (ethyl methanesulfonate) treatment and washing the cells with 0.1 M phosphate buffer (pH 7.0). Screening was carried out on liquid medium and agar medium at a culture temperature of 42 ° C. in YPD medium (yeast extract 10 g, polypeptone 20 g, glucose 20 g, water 1000 mL) containing 1.2 M KCl. Finally, the cells were cultured in a YPD medium containing 20% glucose and 1.2 M KCl, and the BA11 strain was selected using ethanol fermentability (fermentation rate and residual sugar) and agglomeration sedimentation (visual observation of the sedimentation rate) as indices. The BA6 and BA11 strains were grown at 40 ° C in a culture solution prepared by diluting Okinawa molasses (waste molasses from sugarcane recovered three times with sugar) with water and containing about 15% (w / w) sugar. When cultured, it was confirmed that the BA11 strain was superior to the BA6 strain in ethanol fermentability and coagulation sedimentation. Saccharomyces cerevisiae BA11 strain has been deposited internationally at the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation (2-5-8 Kazusa Kamashi, Kisarazu City, Chiba Prefecture, Japan 292-0818) under the accession number NITE BP-793 (Contract date: August 11, 2009).

[Example 2: Production of Saccharomyces cerevisiae BA16 strain]
Saccharomyces cerevisiae BA5 strain, which is excellent in aggregation sedimentation and alcohol resistance, among the strains stored in Bio Academia Co., Ltd., cell fusion of the BA11 strain and the yeast strain having improved aggregation precipitation and ethanol fermentation ability while maintaining the characteristics of the BA11 strain I tried to create. Cell fusion is performed by attaching a separate genetic marker to both strains, fusing with polyethylene glycol, and selecting a fusion strain that complements the genetic marker (Yasuharu Oshima, Molecular genetics experiment method of yeast, Society publication) Center, 1996). The fused strain was cultured at 42 ° C. in a YPD medium containing 1.2 M KCl, and subsequently a colony was formed on a YPD plate medium having the same composition, and a colony with good growth was selected. The selected colony was diluted with water from Okinawa molasses (waste molasses from which sugar was collected three times from sugarcane) and adjusted to contain about 15% (w / w) of sugar at 40 ° C. The BA16 strain having the highest ethanol fermentability was selected by culturing. The BA16 strain was confirmed to maintain the characteristics of the BA11 strain and to be superior to the BA11 strain in ethanol production ability at high temperature and coagulation sedimentation. Saccharomyces cerevisiae BA16 strain has been deposited internationally at the Patent Microorganism Depositary Center for Product Evaluation Technology, Japan (Accession Number NITE BP-794 (2-5-8, Kazusa Kamashi, Kisarazu City, Chiba Prefecture, Japan 292-0818)) (Contract date: August 11, 2009).

[Example 3: Examination of salt tolerance]
Four types of yeasts, BA11 strain, BA16 strain, BA6 strain and S288C strain (standard strain for budding yeast genetics research) were inoculated on YPD agar medium containing no KCl and YPD agar medium containing 2.0 M KCl. And cultured at 35 ° C. for 24 hours.
The results of the YPD agar medium containing 2.0 M KCl are shown in FIG. 1, and the results of the YPD agar medium not containing KCl are shown in FIG. As is apparent from FIGS. 1 and 2, all strains could grow on the YPD agar medium without KCl, but the BA6 strain and the S288C strain could not grow on the YPD agar medium containing 2.0 M KCl. . This result shows that the BA11 strain and the BA16 strain have very excellent salt tolerance.

[Example 4: Examination of fermentation performance of BA11 strain at 40 ° C.]
Okinawa waste molasses (waste molasses recovered from sugar cane 3 times) diluted with water approximately 3 times and adjusted to contain 15% (w / w) sugar and 5-6% (w / w) ash Then, the fermentation performance of the BA11 strain was examined using this diluted molasses dilution as a raw material (see Example 6 for details of the culture method). The results are shown in Table 1.
As apparent from Table 1, the BA11 strain has 7% at a culture temperature of 40 ° C. using a waste molasses diluted solution containing 15% (w / w) sugar and 5-6% (w / w) ash as raw materials. More than v / v) ethanol was produced. This result indicates that the BA11 strain is excellent in heat resistance and ethanol productivity at high temperatures.

[Example 5: Examination of aggregation and sedimentation properties (1)]
The BA11 strain was stirred and cultured in a 200 mL Erlenmeyer flask containing 100 mL of 5% glucose-containing YPD medium for 12 hours under conditions of 35 ° C. and a rotation speed of 150 rpm using a constant temperature shaker. Stirring was stopped and the mixture was transferred to a 100 mL beaker and the state after 1 minute was shown in FIG. As apparent from FIG. 3, the BA11 strain aggregated and settled within 1 minute after stirring was stopped, and the turbidity of the liquid layer disappeared.

[Example 6: Examination of aggregation and sedimentation properties (2)]
The BA11 strain, BA16 strain and S288C strain (standard strain for budding yeast genetics research) were placed in an Erlenmeyer flask containing 5% glucose-containing YPD medium at 35 ° C. and a rotation speed of 150 rpm using a constant temperature shaker. The culture was stirred for 12 hours under the conditions. Each yeast culture was mixed well and transferred to a graduated cylinder and allowed to stand still. Immediately after standing (start) and after 1 minute, photographs were taken to compare sedimentation properties.
The results are shown in FIG. As is apparent from FIG. 4, no sedimentation was observed in the S288C strain. By standing for 1 minute, the turbidity of the liquid layer was not visually observed in both the BA11 strain and the BA16 strain, and the liquid layer and the bacterial cell layer were separated. The BA16 strain showed better sedimentation than the BA11 strain.

[Example 7: Comparison of fermentation performance of BA11 strain and conventional strain A]
Okinawa waste molasses (waste molasses recovered from sugar cane three times) is diluted about 3 times with water to contain about 15% (w / w) sugar and 5-6% (w / w) ash. The adjusted molasses diluted liquid was used as a raw material. The conventional strain A was cultured at 33 ° C., and the BA11 strain was cultured at 38 ° C. to measure ethanol productivity and sugar reduction. Specifically, the BA11 strain was cultured by the following method.
(1) Pre-culture; BA11 strain stored in 15% glycerol in a freezer at −80 ° C. is inoculated with 2 platinum loops in a YPD medium containing 100 ml of 5% glucose in a 500 ml baffled Erlenmeyer flask. The culture was shaken at 170 ° C. for 24 hours. 3 L of 5% glucose-containing YPD medium was placed in a 5 L jar fermenter, and the whole culture solution was put therein, and cultured for 24 hours under conditions of 38 ° C., 200 rpm stirring, and 0.5 VVM aeration.
(2) Main culture: Into a 100-L liquor tank, 60 L of waste molasses (final concentration 0.8% ammonium sulfate added) diluted with water to a sugar content of about 10% is placed, and 3 L of the preculture solution is added to it. Culturing was performed for 24 hours under the conditions of 20 ° C., 20 Hz stirring, and 0.5 VVM aeration. 600 L of diluted molasses having the same composition was placed in a 1000 L culture tank, and 30 L of this cultured bacterial cell solution was placed therein, and cultured for 24 hours under conditions of 38 ° C., 20 Hz stirring, and 0.5 VVM aeration.
(3) Fermentation: Waste molasses diluted in a 10,000L fermenter to a sugar content of about 15% (w / w) and ash content of 5-6% (w / w) (final concentration 0.8% ammonium sulfate added) 4275L was added, and 214L of the above cultured cell solution was put therein, and cultured for 17 hours under the conditions of 38 ° C., slow stirring (150 rpm), and aeration circulation rate of 15 m ³ / hr. After completion of the fermentation, stirring and aeration were stopped, and the cells were clarified for about 2 hours, so that the cells were agglomerated and settled to separate the solid and liquid, and the supernatant was extracted.
(4) Repeated batch fermentation (batch culture); 720 L of sedimented bacterial body fluid in the fermenter was diluted with waste molasses solution (sugar content about 15% (w / w), ash content 5-6% (w / w), final concentration 0. 4500 L (added with 8% ammonium sulfate) was added and fermented under the same conditions as in (3). This operation was repeated.

  The results are shown in FIG. In both the BA11 strain and the conventional strain A (conventional yeast in the figure), the sugar was converted to about 50% ethanol, and an ethanol production yield almost at the theoretical limit was obtained. However, although the data is not shown, since the production yield of the conventional strain A decreased at 34 ° C. and ethanol could not be produced at 38 ° C., the BA11 strain greatly exceeded the conventional strain A in temperature resistance (heat resistance). . In this example, it was confirmed that the BA11 strain could be used for repeated batch culture at 38 ° C. for 8 times. This result shows that BA11 strain is remarkably excellent in ethanol resistance. In the conventional strain A used for comparison, four repeated batch cultures were confirmed at 33 ° C.

[Example 8: Repeated fermentation performance of BA11 strain at 40 ° C]
Okinawa waste molasses (waste molasses recovered from sugar cane three times) is diluted about 3 times with water to contain about 15% (w / w) sugar and 5-6% (w / w) ash. Using the adjusted molasses waste solution as a raw material, the repeated fermentation performance of BA11 strain at 40 ° C. was examined (see Example 6 for details of the culture method).
The results are shown in FIG. From this result, it was confirmed that 4 batch cultures were possible at 40 ° C. using the BA11 strain. The productivity of each batch is 2.33 g / h · L for the first batch, 2.35 g / h · L for the second batch, 2.11 g / h · L for the third batch, and 1.3 g / h · L for the fourth batch. It was 67 g / h · L.

[Example 9: Fermentation performance of BA11 strain and BA16 strain]
Okinawa waste molasses (waste molasses recovered from sugar cane three times) is diluted about 3 times with water to contain about 15% (w / w) sugar and 5-6% (w / w) ash. Using the adjusted molasses waste solution as a raw material, the fermentation performance of the BA11 strain and BA16 strain was examined (see Example 6 for details of the culture method). In the first round, the cells were cultured at 30 ° C. for 21 hours, and the stirring was stopped and the mixture was allowed to stand for 6 hours for aggregation and sedimentation. The supernatant was collected and new raw materials were added. The second incubation was performed at 38 ° C. for 16 hours, and stirring was stopped for 7 hours.
The results are shown in FIG. The BA11 strain used 1 colony (diamond) and the BA16 strain used 3 colonies (circle, triangle, square). As is clear from FIG. 7, the BA16 strain has a fermentation performance equal to or higher than that of the BA11 strain, and it was confirmed that the fermentation performance at a high temperature (38 ° C.) is superior to that of the BA11 strain.

[Example 10: Examination of heat resistance (1)]
The YPD agar medium containing waste molasses diluted about 3 times was inoculated with the BA11 strain and cultured at various temperatures for 24 hours to examine heat resistance. As a control, sake yeast strain K7 (Japan Brewing Association) was used. The results are shown in FIG. As is clear from FIG. 8, the BA11 strain was able to grow even at 43 ° C.

[Example 11: Examination of heat resistance (2)]
The YPD agar medium was inoculated with the BA11 and BA16 strains, and cultured at 38 ° C. or 43 ° C. for 12 hours to examine heat resistance. The S288C strain (standard strain for budding yeast genetics research) was used as a control. The results are shown in FIG. As is clear from FIG. 9, the BA11 and BA16 strains were able to grow even at 43 ° C.

[Example 12: Analysis of ploidy of BA11 strain and BA16 strain]
As comparative strains, the haploid strain SC288C (J. Biol. Chem. 246, 7817-7819. 1971), the diploid strain K14 (Sake Yeast Association No. 14, Japan Brewing Association), and the tetraploid strain SC1333 (also known as NCYC1333) And the BA11 strain and BA16 strain were analyzed for ploidy. Specifically, cells in a logarithmic growth phase cultured at 30 ° C. for 16 hours in a YPD medium were diluted so that OD660 was about 0.1, stained with fluorescent dye propidium iodide (PI), and then flow cytometry. The DNA content per cell and the corresponding number of cells were analyzed by the method.
The results are shown in FIG. In FIG. 10, the horizontal axis of each chart represents the DNA content, and the vertical axis represents the number of cells. As is apparent from FIG. 10, the DNA content distributions of the BA11 strain and BA16 strain were similar to those of the comparative strain K14 (diploid). Therefore, it was confirmed that both the BA11 strain and the BA16 strain are diploid.

[Example 13: Base sequence analysis of rDNA region of BA11 strain and BA16 strain]
The nuclear ribosomal RNA coding region (rDNA) has about 150 copies of repetitive sequences per cell in yeast, and is removed by splicing after transcription. ITS1, ITS2 (Internal Transcribed Spacer 1, 2, see FIG. 11 (A)) Regions have a high rate of evolution and are often different in their own strains within the same species. Therefore, the nucleotide sequence of the ITS region can be used for identification of strains (Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics in “PCR Protocols; A Guide to Methods and Applications”, Academic Press. 1990).
The method for determining the nucleotide sequences of the ITS1 and ITS2 regions of rDNA, the determination of the nucleotide sequences of these regions, and the primer sequences used for cloning were described in Kawahata et al. (Kawahata M. et al. Biosci. Biotechnol. Biochem. 71, 1616). -1620, 2007). S288C strain (haploid), which is a standard strain for budding yeast genetics research, was used as a control. DNAs were extracted from the BA11 strain, BA16 strain and S288C strain, respectively, and the ITS1 and ITS2 region DNAs were amplified by PCR. The obtained DNA fragment was cloned into a plasmid and the nucleotide sequence was analyzed.

The results are shown in FIG. In region A of ITS1, the control strain (S288C strain) has two Ts from the 28th, but the BA11 and BA16 strains are identified as two types of sequences, a sequence with 7 Ts and a sequence with 9 Ts. It was. In region B of ITS1, a sequence in which the 242nd T was replaced with C in the BA11 strain was found in only 1 out of 10 clones, which may be an error caused by PCR. The 301st region of ITS1 region C was C in the control strain, whereas it was T in the BA11 and BA16 strains. In region D of ITS2, the control strain and the BA16 strain had a sequence with 8 Ts, whereas the BA11 strain identified two sequences, a sequence with 8 Ts and a sequence with 9s. In region E, the control strain had a sequence that lasted 7 Ts from the 163rd, whereas the BA11 and BA16 strains had a sequence that lasted 6 Ts.
The fact that the BA11 strain and the BA16 strain have a heterologous sequence region is consistent with the fact that both strains are heterodiploids prepared by the cell fusion method. The BA11 strain and the BA16 strain were found to have at least 4 different sequences in the ITS region from the control strain, and to have unique sequences different from any of the 29 Saccharomyces cerevisiae strains analyzed in the above-mentioned Kawahata et al. Therefore, the base sequence of the ITS region is considered to be extremely effective for distinguishing between the BA11 strain and the BA16 strain.

  The present invention is not limited to the above-described embodiments and examples, and various modifications are possible within the scope shown in the claims, and technical means disclosed in different embodiments are appropriately combined. The obtained embodiment is also included in the technical scope of the present invention. Moreover, all the academic literatures and patent literatures described in this specification are incorporated herein by reference.

Claims (2)

(A) can grow on YPD medium containing 2.0 M KCl;
(B) Using a waste molasses diluted solution containing 15% (w / w) sugar and 5-6% (w / w) ash as a raw material, 7-10% (v / v) ethanol at a culture temperature of 40 ° C. Produce and
(C) After stirring and culturing at 35 ° C. for 12 hours in a 5% glucose-containing YPD medium, the agglomeration settles within 1 minute after stopping the stirring .
(D) Using a waste molasses diluted solution containing 15% (w / w) sugar and 5-6% (w / w) ash as a raw material, repeated batch culture of 8 or more times at a culture temperature of 38 ° C. is possible,
(E) Using a waste molasses diluted solution containing 15% (w / w) sugar and 5-6% (w / w) ash as a raw material, it is possible to perform four or more repeated batch cultures at a culture temperature of 40 ° C. a yeast characterized by comprising:
Yeast which is Saccharomyces cerevisiae BA11 strain (Accession No. NITE BP-793) or Saccharomyces cerevisiae BA16 strain (Accession No. NITE BP-794). An ethanol production method using the yeast according to claim 1 .