Method For Continuous Production Of Alcohol

Abstract

A method for continuous production of an alcohol to produce ethanol from molasses, including a fermentation step of continuously fermenting a mixed liquid containing molasses and ethanol fermentation yeast having flocculating and settling properties in a fermentor to obtain a fermentation liquor; a yeast separation step of continuously withdrawing the fermentation liquor from the fermentor and sending the fermentation liquor to a yeast separation tank, and separating yeast cells of the ethanol fermentation yeast by gravity settling from the fermentation liquor in the yeast separation tank to obtain a de-yeasted fermentation liquor and a yeast cell suspension; a yeast cell return step of continuously withdrawing the yeast cell suspension from the yeast separation tank and returning the yeast cell suspension to the fermentor; and a suspended solids separation step of continuously withdrawing the de-yeasted fermentation liquor from the yeast separation tank, sending the de-yeasted fermentation liquor to a suspended solids separation tank, and separating suspended solids derived from molasses by gravity settling from the de-yeasted fermentation liquor in the suspended solids separation tank to obtain a suspended solids-separated fermentation liquor and a suspension of suspended solids. [Fig-1]

Information

Applicants

Inventors

Specification

FORM 2
THE PATENTS ACT 1970 (39 OF 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)
1. TITLE OF THE INVENTION METHOD FOR CONTINUOUS PRODUCTION OF ALCOHOL
2. APPLICANTS:

i) IBI CHEMATUR (ENGINEERING AND CONSULTANCY) LTD.
AN INDIAN COMPANY
of IBI HOUSE, S 86 ANDHERIKURLA ROAD, CHIMATPADA, MAROL NAKA, ANDHERI (EAST) MUMBAI - 400 059, INDIA
ii) MITSUI ENGINEERING & SHIPBUILDING CO., LTD. A JAPANESE COMPANY of 6-4, TSUKIJI 5-CHOME, CHUO-KU, TOKYO 104-8439, JAPAN
&
iii) CHEMATUR ENGINEERING AB A SWEDISH COMPANY of BOX 430, SE-691,27 KARLSKOGA, SWEDEN
The following specification particularly describes the invention and the manner in which it is to be performed

[TECHNICAL FIELD]
The present invention relates to a method for continuous production of an alcohol. More specifically/ the present invention relates to a method for continuous production of ethanol using molasses as fermentation material.
[BACKGROUND ART]
To prevent global warming, there have been proposed a number of ethanol production processes starting from biomass material, which is carbon-neutral, an example of which is described in Patent Literature 1.
However, the production costs of ethanol incurred when the process starts from biomass material are relatively high as compared with the production costs of fossil fuel such as, for example, gasoline and light fuel. Thus, there are demands for reduced production costs to be met.
Examples of biomass materials currently in use include sugar materials such as sucrose and molasses and starch materials derived mainly from cereals. Among those materials, use of molasses as material for ethanol fermentation is advantageous because of ease of pretreatment.
Methods of fermenting ethanol as biomass material include a continuous fermentation method and a batch fermentation method. The continuous fermentation method is advantageous over the batch fermentation method because of, for example, a reduced reactor volume attained by an improved productivity and reduced personnel costs and a consistent product quality made possible by a simplified operation.
Such process for producing ethanol from molasses by a continuous fermentation method may be exemplified by a process commercialized by Chematur Engineering AB (Sweden) called BIOSTIL (trademark) process.

The BIOSTIL process may be generally broken into a fermentation step, a yeast separation step, a yeast cell return step, a crude distillation step, a de-ethanoled fermentation liquor return step, a rectification step, and a dehydration step. This process may in essence be represented by a flowchart of Fig. 9. The BIOSTIL process features the yeast cell return step of returning yeast cells separated from the fermentation liquor in the yeast separation step to the fermentation step and a de-ethanoled fermentation liquor return step of returning a part of the fermentation liquor (de-ethanoled fermentation liquor) from which ethanol produced in the crude distillation step has been removed by distillation to the fermentation step. The process aims at reducing the running costs and initial costs by recycling the yeast cells and the de-ethanoled fermentation liquor.
[CITATION LIST] [PATENT LITERATURE]
Patent Literature 1: International Publication WO 86/ 003514
[SUMMARY OF THE INVENTION] [TECHNICAL PROBLEMS]
As an ethanol production method starting with molasses, the BIOSTIL process boasts a high processing efficiency, reduced risk of contamination and a high energy yield, achieving reduction in ethanol production costs.
However, the inventors of the present invention found that the above process involves higher running costs and the higher initial capital costs of the centrifugal separator for separating the yeast from the fermentation liquor. The associated mechanical damage to the yeast cells in the centrifugal separators is also likely to reduce the ethanol fermentation efficiency.
To overcome these limitations, the present inventors considered using in the BIOSTIL process a flocculating yeast strain Saccharomyces cerevisiae AMI 2 (which may hereinafter

be referred to simply as "AM12") described in JP 59-135896 A as fermentation yeast and separating the yeast cells from the fermentation liquor by gravity settling.
However, as we considered using a settling and separating tank capable of separating virtually only the yeast cells from the fermentation liquor (a settling and separating tank or a yeast separation device described in WO 08/120644) on the one hand and a settling and separating tank capable of separating both the yeast cells and suspended solids other than the yeast cells from the fermentation liquor (tilted plate settler) on the other hand as a settling and separating tank for separating the yeast cells from the fermentation liquor using the AM12 as fermentation yeast, it was found that in both of the above two cases, the efficiency with which the yeast cells were separated from the fermentation liquor decreased as the continuous fermentation time increased (i.e. as fermentation proceeded further), until finally separation of the yeast cells from the fermentation liquor was no longer possible.
Thus, an object of the present invention is to provide a method for continuous production of an alcohol capable of consistently separating yeast cells from a fermentation liquor by gravity settling using ethanol fermentation yeast having flocculating and settling properties as fermentation yeast in the method, characterized by recycling of yeast cells, for continuous production of an alcohol to produce ethanol from molasses.
[SOLUTION TO PROBLEMS]
Through a study for achieving the above object, the present inventors found that a method for continuous production of an alcohol comprising: an ethanol fermentation step through continuous supply of molasses; a yeast separation step, following the fermentation step, of separating the ethanol fermentation yeast by gravity settling from a fermentation liquor containing the ethanol fermentation yeast having flocculating and settling properties and suspended solids derived from molasses; and a suspended solid separation step, following the yeast separation step, of separating suspended solids by gravity settling from the fermentation liquor from which the ethanol fermentation yeast

has been separated; wherein the ethanol fermentation liquor separated in the yeast separation step is returned to the fermentation step, can provide a method for continuous production of an alcohol capable of consistently separating yeast cells from a fermentation liquor through gravity settling using ethanol fermentation yeast having flocculating and settling properties as fermentation yeast in an alcohol production method for producing ethanol from molasses characterized by the recycling of yeast cells, and thus completed the present invention.
Specifically, the present invention provides the following (1) to (12).
(1) A method for continuous production of an alcohol to produce ethanol from molasses,
comprising
a fermentation step of continuously fermenting a mixed liquid containing molasses and ethanol fermentation yeast having flocculating and settling properties in a fermentor to obtain a fermentation liquor,
a yeast separation step of continuously withdrawing the fermentation liquor from the fermentor and sending the fermentation liquor to a yeast separation tank, and separating yeast cells of the ethanol fermentation yeast by gravity settling from the fermentation liquor in the yeast separation tank to obtain a de-yeasted fermentation liquor and a yeast cell suspension,
a yeast cell return step of continuously withdrawing the yeast cell suspension from the yeast separation tank and returning the yeast cell suspension to the fermentor, a suspended solids separation step of continuously withdrawing the de-yeasted fermentation liquor from the yeast separation tank, sending the de-yeasted fermentation liquor to a suspended solids separation tank, and separating suspended solids derived from molasses by gravity settling from the de-yeasted fermentation liquor in the suspended solids separation tank to obtain a suspended solids-separated fermentation liquor and a suspension of suspended solids.
(2) The method for continuous production of an alcohol according to (1) above, further
comprising

a crude distillation step of continuously withdrawing and sending the suspended solids-separated fermentation liquor from the suspended solids separation tank to an analyzer, distilling the suspended solids-separated fermentation liquor in the analyzer to produce crude distillation ethanol vapors, and liquefying the crude distillation ethanol vapors with a condenser to obtain crude distillation ethanol,
wherein the analyzer comprises a first distillation section (top section) for distilling ethanol from the suspended solids-separated fermentation liquor and a second distillation section (bottom section) for distilling ethanol from the bleed stream containing suspended solids and some yeast separated in the suspended solids separation tank along with a part small stream of de-ethanoled fermentation liquor produced in the first distillation section (top section), wherein both sections are separated by a section separating tray.
(3) The method for continuous production of an alcohol according to (2) above, further comprising a de-ethanoled fermentation liquor return step of withdrawing and returning at least a part of the de-ethanoled fermentation liquor from the analyzer to the fermentor.
(4) The method for continuous production of an alcohol according to (2) or (3) above, further comprising
a rectification step of rectifying the crude distillation ethanol to obtain rectified ethanol,
and
a dehydration step of dehydrating the rectified ethanol to obtain refined ethanol.
(5) The method for continuous production of an alcohol according to any one of (1) to (4) above, further comprising a discharge step of withdrawing the suspension of suspended solids from the suspended solids separation tank and discharging the suspension of suspended solids after concentrating the suspension of suspended solids.
(6) The method for continuous production of an alcohol according to any one of (1) to (5) above,
wherein a surface area A (m2) of the yeast separation tank is in a range of (Q/VYST)
The ethanol fermentation yeast having flocculating and settling properties (which may hereinafter be referred to simply as "flocculating yeast") needs to have both flocculating and settling properties and ethanol fermentation properties but is otherwise not specifically limited.
«Flocculating and Settling Properties»
The flocculating and settling properties herein refer to characteristics (flocculating

properties) such that the yeast cells flocculate and to the characteristics (settling properties) such that SV30 settles faster than the type strain of the Saccharomyces cerevisiae or wild-type strains derived therefrom (hereinafter referred to as "conventional yeast"). Examples of the conventional yeast include JCM 7255, NBRC 10217, DSM 70449, and ATCC 18824 (JCM:RIKEN BioResource Center, NBRQBiological Resource Center, National Institute of Technology and Evaluation, DSM: Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, ATCC: American Type Culture Collection).
The flocculating properties may be evaluated by, for example, feeding 1L of yeast culture solution containing 0 % by mass of solids other than yeast cells into a 1L measuring cylinder and measuring the cell count in the depth direction of the culture solution both upon placement of the measuring cylinder to stand and 30 minutes later. More specifically, the flocculating properties may be evaluated by measuring the cell count near the surface of the culture solution and the cell count at the bottom of the container. In the case of the above flocculating yeast, for example, the cell count near the surface of the culture solution preferably decreases, 30 minutes after the culture solution is placed to stand, to about 2.0 % or less, more preferably about 1.8 % or less, still more preferably 1.6 % or less, and yet still more preferably about 1.5 % or less, of the cell count immediately after the placement of the container, whereas the cell count at the bottom of the container preferably increases, 30 minutes after the culture solution is placed to stand, to about 3.0-fold, more preferably about 3.2-fold, still more preferably about 3.5-fold, and yet still more preferably about 3.6-fold, of the cell count immediately after the placement thereof.
The settling properties may be evaluated using, for example, SV30, a settled sludge volume. SV30 may be obtained by, for example, feeding a 1000 mL yeast culture solution into a 1L measuring cylinder and measuring the ratio (%) of the volume of the yeast cells that have settled 30 minutes after the measuring cylinder is placed to stand. Specifically, when the interface between the settled yeast cells and the supernatant lies at 300 mL, for example, SV30 = 300mL/1000mL x 100 = 30 %. For the above flocculating yeast, SV30 is preferably about 5.0 % or less, more preferably about 4.5 % or less, still more preferably about 4.3 % or less, and yet still more preferably about 4.1 % or less.

In the present invention, the flocculating yeast satisfies the following in the yeast settler:
Q/VYST < A < Q/Vss, and preferably,
Q/VAMI2< Q/VYST
Any fermentor as appropriate known in the art may be used for the invention.
The fermentor preferably stirs the mixed liquid for fermentation containing flocculating yeast and molasses in order to increase the chance of contact between flocculating yeast and molasses.
In order to increase the ethanol productivity of the flocculating yeast, the dilution ratio D (h-1) is preferably 0.03 < D < 0.3, more preferably 0.1 < D

The yeast separation tank that may be used in the invention is required to be capable of causing the yeast cells of the flocculating yeast in the fermentation liquor to separate and settle by gravity but otherwise not specifically limited and is preferably the yeast separating device described in WO 08/120644 allowing the yeast cells of the flocculating yeast to flocculate and settle using the flocculating and settling ability of the flocculating yeast while ensuring that the suspended solids derived from molasses (which may herein be referred to as "SS") are not allowed to settle.

The condenser is used to cool the ethanol vapors and obtain liquid ethanol. The condenser may be of a single stage or of a multi-stage type. A low-temperature heat source is not specifically limited and may be the fermentation liquor supplied to the analyzer from a viewpoint of efficient use of heat energy.

When desired, the method of the invention may further comprise, in addition to the crude distillation step, a de-ethanoled fermentation liquor return step.
6. De-ethanoled Fermentation Liquor Return Step
In the de-ethanoled fermentation liquor return step, the de-ethanoled fermentation liquor is withdrawn from the analyzer, and at least a part thereof is returned to the fermentor.
Inclusion of the above step in the method of the invention allows at least a part of the de-ethanoled fermentation liquor to be fed back to the fermentor and thus reduces not only the amount of water discharged in the crude distillation step but the amount of water supplied in the fermentation step as well.
Further, use of a heat exchanger enables transfer of heat energy of the de-ethanoled fermentation liquor withdrawn from the analyzer to the suspended solids-separated fermentation liquor supplied to the analyzer, simultaneously achieving cooling of the de-ethanoled fermentation liquor and heating of the suspended solids-separated fermentation liquor.
The continuous ethanol production method of the invention may further include, when desired, a step of producing refined ethanol from crude distillation ethanol.
The method of producing refined ethanol from crude distillation ethanol is not specifically limited and may be a method whereby, for example, crude distillation ethanol is further refined by the rectifier to obtain rectified ethanol, followed by dehydration through a dehydrator, to obtain refined ethanol.
To be more specific, one may use, for example, a method as used in the BIOSTIL process whereby rectified ethanol vapor is generated in a rectifier, followed by dehydration through a molecular sieve and condensation, to obtain liquid refined ethanol.

The present invention further provides an apparatus for continuous production of an alcohol for producing ethanol from molasses.
The apparatus for continuous production of an alcohol according to the invention comprises a fermentor for fermenting molasses in the presence of a flocculating yeast to produce a fermentation liquor, a yeast separation tank for separating yeast cells of the flocculating yeast by gravity settling from the fermentation liquor to produce a de-yeasted fermentation liquor where the flocculating yeast has a low yeast cell concentration and a yeast cell suspension where the flocculating yeast has a high yeast cell concentration, and a suspended solids separation tank for separating suspended solids derived from molasses from the de-yeasted fermentation liquor by gravity settling to produce a suspended solids-separated fermentation liquor having a low concentration of the suspended solids and a suspension of suspended solids, wherein the fermentor and the yeast separation tank are connected by a line for delivering the fermentation liquor, wherein the fermentor and the yeast separation tank are connected by a line for delivering the yeast cell suspension, and wherein the yeast separation tank and the suspended solids separation tank are connected by a line for delivering the de-yeasted fermentation liquor.
The apparatus for continuous production of an alcohol may further comprise an analyzer for vaporizing ethanol from the suspended solids-separated fermentation liquor to produce a crude distillation ethanol and a condenser for liquefying the crude distillation ethanol vapors to produce a liquid crude distillation ethanol, wherein the suspended solids separation tank and the analyzer may be connected by a line for delivering the suspended solids-separated fermentation liquor, wherein the analyzer and the condenser may be connected by a line for passing the crude distillation ethanol, and wherein the analyzer may include a first distillation section for distilling ethanol from the suspended solids-separated fermentation liquor and a second distillation section for distilling ethanol from purge stream from SS separator bottom alongwith a de-ethanoled fermentation liquor produced in the first distillation section.

In the apparatus for continuous production of an alcohol according to the invention, the analyzer and the fermentor may be connected by a line for delivering the de-ethanoled fermentation liquor from the analyzer to the fermentor.
The apparatus for continuous production of an alcohol may further comprise a rectifier for producing rectified ethanol vapors having a high ethanol concentration and a rectification residual liquid having a low ethanol concentration by distilling crude distillation ethanol, a dehydrator for dehydrating the rectified ethanol vapors to obtain dehydrated ethanol vapors, and a condenser for liquefying the dehydrated ethanol vapors to obtain refined ethanol, wherein the condenser for liquefying the crude distillation ethanol vapors to obtain crude distillation ethanol and the rectifier are connected by a line for delivering the crude distillation ethanol, wherein the rectifier and the dehydrator are connected by a line for delivering rectified ethanol vapors, and wherein the dehydrator and the condenser for liquefying the dehydrated ethanol vapors to obtain refined ethanol are connected by a line for delivering dehydrated ethanol.
Examples of the process of producing ethanol according to the invention will now be described in greater detail referring to Fig. 2. The method for continuous production of alcohol according to the invention is not limited in any manner to the examples given below and allows various modifications thereof by those skilled in the art.
[Fermentation Step]
Molasses (51) is supplied to a molasses supplier (1) for temporary storage.
The molasses (51) is continuously withdrawn from the molasses supplier (1) and mixed with dilution water (81) to produce a solution of molasses (52).
The solution of molasses (52) is continuously supplied to a fermentor (2). *
In the fermentor (2), the molasses is continuously fermented in the presence of flocculating yeast to produce a fermentation liquor (53).

[Yeast Separation Step]
The fermentation liquor (53) is continuously withdrawn from the fermentor (2) and supplied to a yeast separation tank (3).
In the yeast separation tank (3), the yeast cells of the flocculating yeast are separated by settling to produce a de-yeasted fermentation liquor (54) having a low yeast cell concentration and a yeast cell suspension (55) having a high yeast cell concentration.
3. Yeast Cell Return Step
The yeast cell suspension (55) is continuously withdrawn from the yeast separation tank (3) and continuously returned to the fermentor (2).
[Suspended Solids Separation Step]
The de-yeasted fermentation liquor (54) is continuously withdrawn from the yeast separation tank (3) and continuously supplied to the suspended solids separation tank (4).
In the suspended solids separation tank (4), the suspended solids derived from the molasses (51) in the de-yeasted fermentation liquor (54) are separated by settling to obtain a yeast/SS-separated fermentation liquor (57) having a low concentration of suspended solids and an SS suspension (58) having a high concentration of suspended solids.
[Crude Distillation Step]
The yeast/SS-separated fermentation liquor (57) is continuously withdrawn from the suspended solids separation tank (4) and supplied to the upper section of the analyzer (7), which is a continuous distillation column.

In the analyzer (7), steam (66) obtained by boiling a crude distillation residual liquid (64) with a reboiler (10) is used as a heat source for distillation.
(First Distillation Stage) The yeast/SS-separated fermentation liquor (57) is fed to the upper section of the first distillation section and allowed to move downward inside the analyzer as ethanol is vaporized to produce the first distillation residual liquid (de-ethanoled fermentation liquor (67)). Part of the de-ethanoled fermentation liquor (67) is supplied to the second distillation section as second distillation section-destined supply fermentation liquor (68), and the remainder thereof is returned to the fermentor (2). The second distillation section supplies an ethanol-containing steam (71).
(Second Distillation Stage) The de-ethanoled fermentation liquor (67) is supplied from the upper section of the second distillation section as the second distillation section-destined supply fermentation liquor (68) together with part of the SS suspension (58) and allowed to move downward inside the analyzer as ethanol is vaporized to produce a second distillation residual liquid (crude distillation residual liquid). The second distillation section supplies an ethanol-containing steam (71) to the first distillation section.
(Condensation Stage) The crude distillation ethanol vapors (59) vaporized from the yeast/SS-separated fermentation liquor (57) is introduced from the upper section of the analyzer (7) into the condensers (8) and (9) to produce a waste gas (69) and liquid crude distillation ethanols (61) and (62).
The crude distillation ethanol (61) and the crude distillation ethanol (62) are mixed and stored in a crude distillation ethanol tank (11).
[De-ethanoled Fermentation Liquor Return Step]
Part of the de-ethanoled fermentation liquor (67) withdrawn from the analyzer (7) is supplied to the second distillation section of the analyzer (7), and the remainder thereof is returned to the fermentor (2).

[Discharge of Crude Distillation Wastes]
The crude distillation residual liquid (64) is withdrawn from the bottom section of the analyzer (7), boiled by the reboiler (10), and returned to the bottom section of the analyzer (7). Part of the steam (66) is cooled, liquefied, and mixed with the crude distillation residual liquid (64) reservoired in the bottom section of the analyzer (7). A crude distillation residual liquid where contamination has advanced after the above recycling is repeated is discharged as crude distillation wastes (65).
[Discharge of Suspension of Suspended Solids]
The SS suspension (58) is withdrawn from the suspended solids separation tank (4), part thereof is supplied to the second (bottom) distillation section of the analyzer (7) as the second distillation section-destined supply fermentation liquor (68) together with the de-ethanoled fermentation liquor (67), and the remainder thereof is returned to the fermentor (2).
In the second distillation section of the analyzer (7), the second distillation section-destined supply fermentation liquor (68) containing the SS suspension (58) is further distilled, and the residual liquid thereof is withdrawn as crude distillation residual liquid (64) from the bottom section of the analyzer (7), boiled by the reboiler (10), and returned to the bottom section of the analyzer (7) as a steam (66). Part of the steam (66) is cooled, liquefied, and mixed with the crude distillation residual liquid (64) reservoired in the bottom section of the analyzer (7). A crude distillation residual liquid (64) where concentration of the suspended solids derived from the SS suspension (58) has advanced after the above recycling is repeated is discharged as the crude distillation wastes (65).
Examples

[Example 1]
In this example, crude distillation ethanol was produced from molasses through an ethanol producing process illustrated in Fig. 2.
When the yeast cell separation efficiency has reached a constant state and no longer decreases therefrom after a continued ethanol production, the flocculating and settling ability of the yeast may be regarded as stable so that continuous production of ethanol is possible.
The yeast cell separation efficiency in the yeast separation tank (3) was obtained from the following equation.
Separation efficiency = (amount of yeast cells recovered from fermentation liquor (55) returned from yeast separation tank (3)/amount of yeast cells recovered from fermentation liquor (53) withdrawn from the fermentor (2)
Fig. 4 illustrates a graph plotting the continuous fermentation time on the horizontal axis against the separation efficiency on the vertical axis.
While there is a variation, the separation efficiency may be considered stable, with most measurements thereof settling above 0.7.
To observe the degree of yeast deterioration, comparison was made between the yeast cell count at the start of continuous fermentation (at 8th h in Fig. 4) and the yeast cell count at the end of continuous fermentation (at 116th h in Fig. 4). The comparison showed that where OD600 = 1, the yeast cell count was 9.7 x 107 at the start of the continuous fermentation, while it was 8.5 x 107 at the end of the continuous fermentation. Thus, the yeast that was separated and recovered from the fermentation liquor exhibited only a small degree of deterioration.

It follows, therefore, that the ethanol producing process according to the example of the present invention illustrated in Fig. 2 enables continuous production of ethanol because the yeast cell separation efficiency may be considered stable and, hence, the amount of yeast cells returned from the yeast separation tank to the fermentor may also be considered stable.
[Comparative Example 1]
In this comparative example, crude distillation ethanol was produced from molasses through an ethanol producing process illustrated in Fig. 3.
When the yeast cell separation efficiency has reached a constant state and no longer decreases therefrom in a continued ethanol production, the flocculating and settling ability of the yeast may be regarded as stable so that continuous production of ethanol is possible. When the yeast cell separation efficiency continuously decreases, the flocculating and settling ability of the yeast may be regarded as decreasing, so that production of ethanol will finally stop.
The yeast cell separation efficiency in the yeast separation tank (3) was obtained from the following equation.
Separation efficiency = (amount of yeast cells recovered from fermentation liquor (55) returned from yeast separation tank (3)/amount of yeast cells recovered from fermentation liquor (53) withdrawn from the fermentor (2)
Fig. 5 illustrates a graph plotting the continuous fermentation time on the horizontal axis against the separation efficiency on the vertical axis.
While there is a variation, the separation efficiency generally tends to decrease and will decrease to 0.5 or less after a continuous fermentation of about 10 hours.

It follows, therefore, that because the yeast cell separation efficiency decreases and the yeast cells will in due course cease to be returned from the yeast separation tank to the fermentor, the ethanol producing process according to the comparative example in the present invention illustrated in Fig. 3 will become unable to produce ethanol continuously.
[Test Example 1]
A study was made to consider the effects of the suspended solids derived from the molasses (referred to below simply as "SS" in this test example) on the flocculating and settling ability of the flocculating yeast strain Saccharomyces cerevisiae AM12 (referred to below simply as "AM12" in this test example).
(Test 1) Study of Effects of SS Concentration on Flocculating and Settling Ability of AM12
The test was conducted to evaluate the flocculating and settling ability of the AM12 under conditions that the AM12 has a constant yeast cell concentration while the SS concentration varies.
(1) Preparation of Yeast Cell Suspension
The AMI 2 was cultured in a 20 % by mass solution of molasses (amount of SS = 0.5 % by mass to 1 % by mass). Next, the culture solution was centrifuged, and settled yeast cells were recovered. The recovered yeast cells were washed with a 20% by mass solution of molasses and centrifuged to recover settled yeast cells.
A 20% by mass solution of molasses was centrifuged to recover settled SS.
The recovered SS was redispersed in a 20% by mass solution of molasses to prepare a 20% by mass solution of molasses having a 5-fold SS concentration (amount of SS = 2.5 to 5 % by mass) and a 20% by mass solution of molasses having a 10-fold SS concentration (amount of SS = 5 to 10 % by mass).

Recovered yeast cells were redispersed in a 20% by mass solution of molasses, a 20% by mass solution of molasses having a 5-fold SS concentration, and a 20% by mass solution of molasses having a 10-fold SS concentration to an OD600 of 26 to obtain yeast cell dispersions. The obtained dispersions will be referred to as normal SS concentration dispersion, 5-fold SS-concentration dispersion, and 10-fold SS-concentration dispersion, respectively, in this test example.
(2) Measurement of Flocculating and Settling Ability
The normal SS concentration dispersion, the 5-fold SS-concentration dispersion, and the 10-fold SS-concentration dispersion were respectively placed in acrylic settling tubes having a diameter of 5 cm and a height of 50 cm to a height of 50 cm, and the tops were covered with a parafilm.
The height of the interface between the settling slurry portion and the clear portion was measured with respect to time, counting from 0 minute, at which the tops were closed with the parafilm.
The graph in Fig. 6 shows the measurement results plotting the standing time (minutes) on the horizontal axis against the height (cm) of the interface between slurry and clear portion on the vertical axis (•: normal SS concentration dispersion, ODeoo = 26; o: 5-fold SS concentration dispersion, ODeoo = 26; x: 10-fold SS concentration dispersion, OD600 = 26).
(3) Measurement of Flocculating and Settling Ability
Fig. 6 shows that as the SS concentration in the suspension increases, the height of the interface between slurry and clear portion of the suspension descends at a lower velocity, that is, the flocculating and settling ability of the AM12 decrease.
It is inferred that the flocculating and settling ability of the AM12 decreased because SS inhibited the flocculation of the AMI 2, restraining floe formation.

(Test 2) Study of Effects of Yeast Cell Concentration on Flocculating and Settling Ability of the AM12
The test was conducted to evaluate the flocculating and settling ability of the AM12 under conditions that SS has a constant concentration (amount of SS = 0.5 to 1 % by mass) while the yeast cell concentration varies.
Preparation of Yeast Cell Suspension
The AM12 was cultured in a 20 % by mass solution of molasses (amount of SS = 0.5 to 1 % by mass). Next, the culture solution was centrifuged, and settled yeast cells were recovered. The recovered yeast cells were washed with a 20% by mass solution of molasses and centrifuged to recover settled yeast cells. The yeast was redispersed in a 20% by mass solution of molasses to obtain yeast cell dispersions each with OD600 = 26, OD600 = 63, and OD600 = 132.
A control dispersion was prepared by allowing the yeast cells of the laboratory yeast strain Saccharomyces cerevisiae S288C to suspend in a 20% by mass solution of molasses sothatOD600 = 26.
(2) Measurement of Flocculating and Settling Ability
The yeast cell dispersions each with OD600 = 26, OD600 = 63, and OD600 = 132 were respectively poured into acrylic settling tubes having a diameter of 5 cm and a height of 50 cm to a height of 50 cm, and the tops were closed with a parafilm.
The height of the interface between the settling slurry portion and the clear portion was measured with respect to time, counting from 0 minute, at which the tops were closed with the parafilm.

The graph in Fig. 7 shows the measurement results plotting the standing time (minutes) on the horizontal axis against the height (cm) of the interface between slurry and clear portion on the vertical axis (•: AM12, OD600 = 26; ▲: AM12, OD600 = 63; ■: AM12, OD600 = 132; and o: S288C strain, OD600 = 26).
(3) Evaluation of Flocculating and Settling Ability
Fig. 7 shows that as the yeast cell concentration in the suspension increases, the height of the interface between slurry and clear portion of the suspension descends at a lower velocity, that is, the flocculating and settling ability of the AM12 decrease.
It is inferred that the flocculating and settling ability of the AMI2 decreased because physical interference between yeast cells resulted in a hindered settling.
It is apparent that the AM12 has excellent flocculating and settling ability over the strain S288.
(Test 3) Study of Effects of SS Concentration on SS Settling Separation Behavior
This test example was conducted to study the SS settling separation behavior in the absence of yeast cells as the SS concentration varies.
(1) Preparation of SS Dispersion
A 20% by mass solution of molasses (amount of SS = 0.5 % by mass to 1 % by mass; major constituents of SS were S and Ca) was centrifuged to recover settled SS. The recovered SS was redispersed in a 20% by mass molasses to prepare an SS dispersion having a 2.5-fold SS concentration (amount of SS = 1.25 to 2.5 % by mass) and an SS dispersion having a 5-fold SS concentration (amount of SS = 2.5 to 5 % by mass).

(2) Observation of Settling Separation Behavior
A 20% by mass solution of molasses having a normal SS concentration (amount of SS = 0.5 to 1 % by mass) and SS dispersions each having a 2.5-fold SS concentration (amount of SS = 1.25 to 2.5 % by mass) and 5-fold SS concentration (amount of SS = 2.5 to 5 % by mass) were respectively poured into acrylic settling tubes having a diameter of 5 cm to a height of 50 cm, and the tops were closed with parafilm.
The height of the interface between the settling slurry portion and the clear portion was measured with respect to time, counting from 0 minute, at which the tops were closed with parafilm.
The graph in Fig. 8 shows the measurement results plotting the standing time (minutes) on the horizontal axis against the height (cm) of the interface between slurry and clear portion on the vertical axis (•: normal SS concentration, ▲: 2.5-fold SS concentration, and ■: 10-fold SS concentration).
(3) Study of Settling Separation Behavior
Fig. 8 shows that as the SS concentration in the suspension increases, the height of the interface between slurry and clear portion of the suspension lowers at an increased velocity, that is, the SS settling separation velocity increases.
It is inferred that the SS settling separation velocity increased because the increased SS concentration facilitated flocculation of SS particles.
(Test 4) Consideration of Yeast Separation tank Capacity At
From Fig. 7, the settling velocity VAMI2 of the yeast cells of the AM12 in the solution of molasses is 6.0 x 10-4 (m•s-1). From Fig. 8, the settling velocity Vss of SS in the solution of molasses is 1.2 x 10-4 (m s-1).

For substantially only the yeast cells to be separated in the yeast separation tank, the following relations need to hold simultaneously.
v1 = Q1/A1 < VAMI2 and
v1= Q1/A1 > Vss [where V1(m s-1) is the surface loading rate of the yeast separation tank, Qi the amount of inflow into the yeast separation tank, and A1 (m2) is the surface area of the yeast separation tank].
Therefore, A1 needs to satisfy
Ql/VAM12

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