CA2054860A1 - Process for continuously fermenting saccharides to produce alcohol using a flocculating microorganism - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

A PROCESS FOR CONTINUOUSLY FERMENTING TO PRODUCE
ALCOHOL USING A FLOCCULATING MICROORGANISM
There is disclosed a process for continuously fermenting saccharides to alcohol using a flocculating microorganism as an alcohol fermentation microorganism.
The process comprises at least two steps; wherein in the first step, a stirring-type cell propagation tank equipped with or without a respiratory quotient-measuring apparatus, an oxygen supply apparatus, a temperature controller, and a cell separation apparatus is used; the quantity of propagation of cells and the fermentation yield of alcohol are controlled using the respiratory quotient (RQ) as an index, so that a flocculating microorganism substantially high in fermentation activity is continuously grwon, and in the second step, said flocculating microorganism is fed to a fermentation tank for continuously producing highly concentrated alcohol equipped with an oxygen supply apparatus, a temperature controller, and a cell separation apparatus.

Description

20S48~0 A PROCESS FOR CONTINUOUSLY FERMENTING TO PRODUCE
ALCOHOL USING A FLOCCULATING MICROORGANISM

FIELD OF THE INVENTION
The invention relates to a process for continuously producing alcohol by fermenting saccharides using flocculating microorganisms.

BACKGROUND OF THE INVENTION
Generally, when continuous fermentation of saccharides into alcohol is carried out, fermentation micxoorganisms suffer from fermentation inhibition and propagation inhibition by the produced alcohol and the viable cell ratio and the fermentation activity drop in the fermentation tank. Further, the fermentation activity of cells drops in an environment where the alcohol conce'ntration is high, and therefore it is required to keep the alcohol concentration in the fermentation tank to a low level, and a large amount of energy is needed for the concentration and separation in the subsequent step.
Conventionally, it is difficult to operate continuously stably for a long period of time with the ethanol concentration being kept, for example, at 70 g~l or over.
To overcome the above problems, a variety of processes and apparatuses are suggested. That is, it is .
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20~4860 required to increase the cell concentration in the fermentation tank so that the productivity of alcohol by fermentation may be increased, and in order to attain that, for example, there are suggested a process wherein an immobilized yeast is used (Alcohol Handbook published by Zaidan-Hojin Hakko-Kogyo-Kyokai (March 15, 1988), pages 145 to 147), a process wherein cells are supported on a carrier (Alcohol Handbook mentioned above, pages 149 to 150), and a process wherein cells are recovered from the fermented mash by centrifugal separation to be returned to the fermentation tank (Alcohol Handbook mentioned above, page 146). However, these processes are accompanied by such problems as the high cost of the carrier of cells and deterioration of the carrier itself. There is also the problem that the machinery cost of a centrifugal separator and the running cost are high.
' On the other hand, a process for production by fermentation is proposed wherein a flocculating yeast is used, a settling separation tank is provided to a tower-type fermentation tank, and two such tower-t~pe fermentation tanks are arranged in series (Alcohol ~U~}QS mentioned abo~e, pages 150 to 151, and J.
FERMENT. BIOENG., Vol. 69, No. 1, 39 to 45, 1990~, but when the cell concentration is increased in a tower-type fermentation tank, the stirring in the fermentation tank .
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20S48~

blecomes not satisfactory, thereby causing, for example, a deviated flow and the fermentation yield disadvantageously lowers. Also there is the problem that agglomerates of cells are formed, thereby clogging the line for transmitting the liquid.
Further, the process wherein the control of the respiratory quotient (RQ) is applied to the cultivation of cells is known to be effective in feeding culture in the production of baker's yeasts (Seibutsu Hanno Process SYstem Handbook, published by Science Forum (April 25, 1985), pages 238 to 244). In this case, the feeding amount of saccharide is controlled to keep the RQ in the range of 1 to 1.2, so that the propagation of yeast may be kept optimum. However, there is no example in which RQ is used for continuous production of alcohol.

S~RY OF THE INVENTION
Therefore the object of the invention is to provide a process for continuously producing alcohol by fermenting saccharides effectively and stably.
Other and further objects, features, and advantages of the invention will appear more evident from the following description taken in connection with the accompanying drawings.

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20~860 BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 is a flow sheet of one embodiment for practicing the present process; Fig. 2 is a flow sheet of another embodiment for practicing the present invention.

DETAILED DESCRIPTION OF THE INVENTION
The inventors have studied to solve these problems in various ways and have found that, in order to keep the concentration of cells in a fermentation tank high, when fermentation of saccharides into alcohol using a flocculating microorganism is carried out in two steps, and when a stirring-type cell propagation tank equipped with an oxygen supply apparatus and cell separation apparatus is used in the first step, to culture continuously a flocculating microorganism in a high concentration, and when a settling separation apparatus is used'in a fermentation tank of the second step, wherein alcohol is substantially produced continuously, the microorganism high in alcohol fermentation activity can be fed continuously in a fermentation tank of the second step, the operation can be effected continuously stably for a long period of time with a high concentration of alcohol being retained, which has overcome the above problems and has led to the present invention.

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20~4860 That is, according to the present invention, there is provided a process for continuously fermenting saccharides to alcohol using a flocculating microorganism as an alcohol fermentation microorganism, characterized in that the production of alcohol is effected in at least two steps; in the first step, a stirring-type cell propagation tank equipped with an oxygen supply apparatus and a cell separation apparatus is used, so that flocculating microorganisms substantially high in fermentation activity are continuously grown; and said flocculating microorganisms are fed to a fermentation tank in the second step for continuously producing highly concentra~ed alcohol (referred to as a first embodiment).
Further the inventors have studied keenly and have found that, in order to keep the concentration of cells in a fermentation tank high, when (1~ fermentation of s~ccharides into alcohol using a flocculating microorganism is carried out in two steps, and when ~2) a stirring-type cell propagation tank equipped with an oxygen supply apparatus, a temperature controller, and a settling separation apparatus separating and condensing cell~ is used in the first step, and the quantity o~
propagation o~ cells and the alcohol yield are controlled with using the respiratory quotient (RQ) in that cell propagation tan~ as an inde~, a microorganism high in . -. . : :
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, 20~860 flocculability and fermentation activity can be grown in the cell propagation tank in the first step and can continuously be fed quantitatively to a fermentation tank of the second step, and therefore while the microorganism high in alcohol fermentation activity can be kept at a high concentration in a fermentation tank of the second step, the operation can be effected continuously stably for a long period of time with a high concentration of alcohol being retained, which has overcome the above problems and has led to the present invention.
That is, another embodiment of the present in~ention provides a process for continuously fermenting saccharides to alcohol using a flocculating microorganism as an alcohol fermentation microorganism, characterized in that the production of alcohol is effected in at least two steps; in the first step, a stirring-type cell propagation tank'equipped with a respiratory quotient-measuring apparatus, an oxygen supply apparatus, a temperature controller, and a cell separation apparatus is used; the quantity of propagation of cells and the fermentation yield of alcohol are controlled using the respiratory quotient (RQ) as an index, so that a flocculating microorganism substantially high in fermentation activity is continuously grown, and said flocculating micraorganism is fed to a fermentation tank for continuously producing . .

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20~860 highly concentrated alcohol equipped with an oxygen supply apparatus, a temperature controller, and a cell separation apparatus in the second step (referred to as a second embodiment).
In the present process, as the flocculating microorganism utilized in the alcohol fermentation, a yeast or a bacterium whose cell concentration can be increased by using a settling separation apparatus is used. Microorganisms whose propagation is facilitated particularly under aerobic conditions are preferable.
For example, a flocculating yeast of the genus Saccharomyses cerevisiae can be used.
In the present process, by utilizing such flocculating microorganisms, it is possible to keep the concentration of cells at a high level and to obtain the cells inexpensively, leading to a low cost of propagating cel~s. On the other hand, when a non-flocculating microorganism is used, such problems occur that not only are cells not utilized but also the volume of the fermenting tank is required to be large due to the low concentration of cells, because cell~ flow out with the fermented mash.
In the present process, as saccharides, for example glucose, sucrose, fructose, xylose, galactose, cellobiose, starch-saccharified liquid, and a juice of ,, . ~ ,, . , . ' molasses, sugarcane, or sugar beet can be used.
In the first embodiment of the present invention, the most suitable condition for feeding oxygen into the cell propagation tank of the first step may be determined by the kind of microorganism, but preferably oxygen is fed in the rate of 1 to 1,500 mol/m3-hr, preferably 50 to 1,000 mol/m3.hr. In the case of feeding air or air containing ox~gen in a high concentration, air that contains oxygen in an amount corresponding to the above rate i5 fed. The amount of oxygen to be fed into the fermentation tank of the second step is preferably in a range of 0 to 300 mol/m3-hr, meaning that the amount of oxygen can be reduced compared with the first step, because the alcohol fermentation does not require oxygen appreciably, thus it may be 0 according to the kind of microorganism.
' In the second embodiments of the present process, as a respiratory-quotient-measuring apparatus, any respiratory-quotient-measuring-apparatus can be used which can measure the consumption of oxygen ~Qo2) and the amount of produced carbon dioxide (QC02) in the tank placed in the cell propagation tank of the first step, and for example the respiratory-quotient-measuring apparatus may comprises an apparatus for measuring the amounts of gases at the inlet and the outlet of the tank, an 20~4860 apparatus for measuring the concentration of oxygen and carbon dioxide, and, if necessary, an apparatus for c~lculating the respiratory quotient (RQ), RQ being equal to Qc02/Qo2-In the present process, the inventors have studied the propagation of cells in the first step in detail and have ~ound that, at the time of semiaerobic culturing, adenosine triphosphate (ATP) is produced both in the respiration and in the ethanol fermentation, and the cell yield takes a value intermediate between that of only the respiration and that of only the ethanol fermentation, and if in this semiaerobic system it is assumed that (the production of cells~the production of ATP) and (the production of cells/the amount of utilized saccharides) are constant, the cell yield YX/s (g/g) and the ethanol yield Yp/s (mole/mole) per the amount of uti~ized saccharides can be expressed by the RQ, which is an index of the aerobic degree. For example, assuming YX/s at the complete aerobic condition to be 0.5 and YX/s at the complete anaerobic condition to be 0.05, the expressions are as follows:
(3RQ~16)(1-Z) OX -- ( 1 ) {60(RQ-1)(1-ZRED)~38(1-Z )}

38{10(1-zRED~ _zX)}(RQ_l)(l-Z) 3{60(RQ-1)(1-ZRED)+38(1-Z0x)} - (2) ., 20~4860 wherein Z is the number of moles of the saccharide consumed for the production of by-products (e.g., glycerol) without the production of ATP when l mole of a monosaccharide is utilized. If only glycerol is taken into consideration, from the experimental values, zOX (z at the complete aerobic condition) is about 0.05 and zRED ~z at the complete anaerobic condition) is about O.OS. It is considered that Z under semiaerobic conditions takes a intermediate value of these values and is variable depending on RQ, and, for example, calculation is possible on the assumption that it is 0.025. From these, the relationship among the culturing conditions (RQ
and the saccharides-feeding speed) of the first step and the cell concentration and the alcohol concentration of the liquid flowed out of the first step can be estimated to determine the operating conditions of the first step.
' In the present process, although the optimum RQ
of the cell propagation tank of the first step is determined by the kind of cell and the ethanol concentration of the fermentation tank of the second step, desirably the operation is effected with the optimum RQ
being in the range of 2 to 200, preferably in the range of 15 to 100. If air or air containing oxygen in a high concentration is fed, the control of RQ is effected by changing the feed speed of oxygen and the number of 20~860 stirring revolutions. If RQ is too large, the propagation speed of cells lowers. On the other hand, if RQ is too small, the inclination of propagation of cells increases but the flocculating property deteriorates and a high cell concentration cannot be kept.
In the present process, preferably the cell propagation tank of the first step and the fermentation tank of the second step are each provided with an apparatus for dispersing gases as an oxygen-feeding apparatus and an apparatus for controlling the flow rate.
In the present process, the cell propagation tank of the first step is provided with a stirring apparatus for stirring cells at a high concentration and saccharides sufficiently, to control RQ. The type of the lS stirring apparatus can be of any one if the mixing in the tank can be effected satisfactory, and preferably the stir~ing apparatus is equipped with stirring blades small in shearing force, such as propellers and ribbon blades.
This is because if the shearing force is large, it influences adversely the flocculating performance of cells, i.e., in ~ome cases cells settle unsatisfactorily in the cell-settling separation tank and a high concentration of cells cannot be retained.
In the present process, the fermentation tank of ~5 the second step is not necessarily a stirring tank and may ,, .

20S~860 be a tower-type fermentation tank, but an apparatus for mixing in the fermentation tank is required. For example, an air lift-type fermentation tank may be used.
Preferably if a fermentation tank of a stirring tank-type is used, the mixed state can be suitably controlled.
The cell-settling separation tanks used for the fermentation tanks of the first and second steps may be provided inside or outside of the fermentation tanks, and the cell concentration in the fermentation tank may be kept at 10 g/l or more, preferably 50 to 120 g/l, in terms of the weight of dried cells.
Further, the cells propagated in the cell propagation tank of the first step flow from the settling separation tank and they are sent to the fermentation tank of the second step.
In the present process, it is possible that the conce'ntration of saccharides to be fed and the feeding speed of saccharides are changed for the cell propagation tank of the first step and for the fermentation tank of the second step independently, and the concentration of saccharides to be ed to the cell propagation tank Ls pre~erably 1 to 20 wt.~, more prefera~ly 5 to 15 wt.%. On the other hand, the concentration of saccharides to be fed to the fermentation tank of the second step is preferably 20 wt.~ or more, more pr0ferably 20 to 60 wt.%.

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~ 20S4860 The remaining saccharide concentration in the cell propagation tank of the first step is preferably 50 g/l or less, more preferably 10 g/l or less, and the remaining saccharide concentration in the fermentation tank of the second step is preferably 10 g/l or less, more preferably 3 g/l or less. If the concentration is high, the settling and separation of cells becomes unsatisfactory due to the carbon dio~ide given off along with the fermentation in the settling separation ~ank, thereby not only causing the cell concentration in the fermentation tank to lower but also increasing unfermented saccharides that flow out, which lowers the fermentation yield of alcohol.
In the present invention, the alcohol concentration in the cell propagation tank of the first step is preferably 70 g/l or less, more preferably 60 g/l or les. If the alcohol concentration is over 70 g/l, the propagation inhibition becomes large due to alcohol, and not only the cells die to lower the cell concentration, but also alcohol fermentation inhibition takes place, thereby causing the fermentation speed to drop. In the fermentation tank of the second step, the fermentation is effeated so that the alcohol concentration may be 70 g/l or more, preferably 80 to 150 g/l.
In the present invention, the temperature , ' . , "

20~4860 controllers provided to the cell propagation tank of the first step and to the fermentation tank of the second step may be those which can control the temperature in these tanks, for example, by passing water whose temperature has s been adjusted into a jacket provided adjacent to the outside of the tank.
In the present process, the fermentation temperatures in the cell propagation tank of the first step and the fermentation tank of the second step are determined depending on the type of cells and are in the range of 20 to 65C and 15 to 40C, respectively. Since generally at a high temperature the alcohol fermentation activity is inhibited highly, the temperature in the alcohol fermentation tank is desirably controlled to a temperature lower than that in the cell propagation tank by 5 to 15C.
' In the present process, in order to recover alcohol from the fermentation exhaust gas from each fermentation tank, an alcohol-recovering apparatus, such as a condenser or scrubber, can be provided.
In the present process, a solution of a nutrient required for the propagation of cells, such as yeast extract, malt extract, and polypeptone, can be added into the cell propagation tank of the first step and the fermentation tank of the second step.

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-' 20~4860 The invention will now be described in more detail with reference to the drawings.
Fig. 1 is a flow sheet showing one mode of this invention, wherein a saccharide is fed from a line 4 and oxygen, air, or the like is fed from a line 5 into a stirring-type cell propagation tank 1 of a first step.
The propagation of cells of a flocculating microorganism for use in alcohol fermentation is facilitated under aerobic conditions. A nutrient solution for the cell propagation can be fed through a line 4 into the cell propagation tank 1 and through a line 13 into a fermentation tank 2.
The stirring-type cell propagation tank 1 is equipped with a gas-dispersing apparatus for supplying oxygen and stirring blades for stirring cells at a high concentration and sa. The shape of the stirring blades is preferably one which creates a small shearing force, and examples of the stirring blades are propellers and ribbon blades. The fermented liquid in the stirring-type cell propagation tank 1 is passed through a line 22 into a settling separation tank 3 (if a separation tank 3 is provided in the fermenta~ion tank, the lines 22 and 23 are not required).
The liquid separated and flowed out from the set~ling separation apparatus 3 of the cell propagation ^- 20~4860 tank 1 is passed through line 8 and all or a part of the liquid is sent into an alcohol fermentation tank 2 of the second step or into a line 11 through a line 9.
When this flowed out liquid almost does not contain saccharide and alcohol, the liquid is discharged through the line 9 to a line 10.
On the other hand, settled and separated cells are fed into the alcohol fermentation tank 2 in an amount as needed through a line 6, and remainings are sent back into the cell propagation tank 1.

The fermentation gas is discharged through a line 16 of the propagation tank 1 and a line 20 of the settling separation tank 3. Alcohol contained in the fermentation gas is recovered by an alcohol-recovering apparatus 24 through a line 25, and the fermentation gas is discharged from a line 26.
' As the fermentation tank 2 of the second step, for example, an air lift-type fermentation tank can be used. Further, the fermentation gas discharged from a line 14 of the fermentation tank 2 may be passed through lines 15 and 12 to be circulated into the fermentation tank 2, to obtain a stirring effect. Preferably, a stirring tank-type fermentation tank is used, and in that case, the mixed state can be properly controlled. The alcohol fexmentation gas is discharged through the line 14 ~O~8~60 and a line 21. Air or oxygen to the fermentation tank 2 is supplied through the line 12.
The alcohol-fermentation-completed liquid is passed through a line 23 into the settling separation tank 33, where the cells are separated, and the liquid containing cells is returned through a line 17 into the fermentation tank 2. If necessary, the cells can be removed through a line 18 and the removed cells can be sent through a line 19 into the cell propagation tank 1.
The cell-settling separation tanks 3 and 33 may be provided inside or outside of the fermentation tank. The concentration of the saccharide and the feeding speed of the saccharide can be changed for the propagation tank 1 and the fermentation tank 2 independently. It is preferable that the concentration of the saccharide to be fed through the line 13 to the fermentation tank 2 is 20%
or mo~e.
Further, the operation is carried out in such a way that the concentration of the saccharide in the cell propagation tank 1 and the fermentation tank 2 is 50 g/l or less, preferably 10 g/l or less.
Fig. 2 is a flow sheet showing another mode of this invention. This flow sheet is the same as Fig. 1, except that the cell propagation tank 1 of the first step is equipped with a respiratory quotient-measuring 20~860 apparatus 27 and lines 29 and 30, between lines 5 and 16 f and a line 28 from the cell-settling tank 3 is connected to the line 16 from the cell propagation tank 1 at the point before the line 30 being connected.
The propagation of cells of a flocculating microorganism for use in alcohol fermentation is, as described above, facilitated under aerobic conditions.
The cell yield and the alcohol fermentation yield have an interrelationship with the respiratory quotient (RQ). The control of RQ can be effected such that RQ is kept at a prescribed value by measuring the gas flow rates of the line 5 and 16 and the oxygen concentration and the carbon dioxide concentration by an RQ-measuring apparatus 27, and then by using the value of RQ based on the measured values, the supply of oxygen and the number of stirring revolutions are controlled. Preferably this control can be ef'fected in an on-line manner, but the control may be effected in an off-line manner.
The stirring-type cell propagation tank 1 is equipped with an oxygen supply apparatus and a stirring apparatus for RQ control.
Reference numbers in Fig. ~ identical with Fig.
1 indicate matters identical with Fig. 1, and have working functions identical with Fig. 1, so duplicated descriptions are omitted. In Fig. 2, lines may be .

205~860 provided for discharging the liquid flowed out from the settling separation apparatus 3, when the liquid almost does not contain saccharide and alcohol.
Now the present process will be described in more detail with reference to an Example and Comparative Examples.

ExamPle 1 As saccharides, Philippine molasses containing 56.6% of fermentable saccharide was used. The composition of the fermentation culture medium was such that it contained as a nutrient source 3 g/l of ammonium sulfate, and as an antifoaming agent 0.1 ml/l of Adekanol LG 294 (trade name). The Philippine molasses was diluted with water so that the saccharide content for the cell propagation tank might be adjusted to 80 g/l and the saccharide content for the alcohol fermentation tank might be 30'0 g/l.
The fermentation mediums were sterilized at 120C for 10 min and were fed to the respective fermentation tanks.
As a flocculating microorganism, Saccharomyses cerevisiae IR-2 was used, and the ethanol fermentation was carried out according to Fig. 1. This flocculating microorganism was preliminarily grown in a usual manner before being introduced into the present process.

`~ 2~860 Through the line 4, the molasses medium having a saccharides content of 80 g/1 was introduced into the cell propagation tank 1 at such a flow rate that the dilution rate was 0.187 h-l. At that time, 10% of the liquid in the cell propagation tank was inoculated. The supply of oxygen was carried out by passing air into the cell propagation tank. The feeding rate of exygen was set at 140 mo/m3-hr. The stirring blades were of the propeller type and the number of rotations was 200 rpm. The fermentation temperature was 30C and the pH was kept at 4.75 to 4.8.
At that time, in the cell propagation tank 1, the cell concentration became 120 g/l in terms of the dry weight, the alcohol concentration became 46 g/l, and the remaining saccharides concentration became 0.1 g/l or less. This fermentation liquid was introduced into the sett~ing separation tank 3 and all of the liquid flowed out therefrom was introduced into the fermentation tank 2.
At that time the cell concentration of the liquid flowed out was 13 g/l. All of the separated cells were returned to the cell propagation tank 1.
On the other hand, the molasses medium having a saccharldes concentration of 300 g/l was supplied to the fermentation tank 2 with the dilution rate being 0.075 h-1, and as a supply of oxygen, air was used with a ~, :

:' 20~860 feeding speed of 22.3 mol~m3-hr. The stirring blades were of the propeller type and the number of rotations was 100 rpm. The fermentation temperature was 30C and the pH was kept at 4.75 to 4.8.
At that time, in the fermentation tank 2, the cell concentration could be kept at 90 to 100 g/l and the alcohol concentration could be kept at as high as 80 g/l.
The remaining saccharides concentration became 3 g/l or less.
The dilution rate to the whole fermentation tank volume in this test became 0.118 h-1. Under these operating conditions, the operation was possible stably and continuously for 500 hours or more.
Comparative ExamPle 1 lS An alcohol fermentation test was performed using only one tank, which was the fermentation tank 2. The ferméntation medium was the same molasses medium that was used in Example 1. The saccharide concentration was 180 g/l and the dilution rate was the same as that to the whole fermentation tank volume of Example 1. Other fermentation tank conditions were the same as those in Example 1.
As a xesult, the cell concentration in the fermentation tank 2 gradually decreased to 30 g/l and the alcohol concentration also decreased, so that stable -- 20~860 operation was impossible.
When fermentation was performed similarly using only one tank with the cell concentration being 300 g/l and the dilution rate being 0.05 h-l, the remaining saccharides concentration became 100 g/l or more and settling and separation became difficult, so that stable operation was impossible.
Example 2 As a saccharide, Philippine molasses containing 51.5% of fermentable saccharide was used. The composition of the fermentation culture medium was such that it contained as a nutrient source 2.5 g/l of ammonium sulfate, and as an antifoaming agent 0.1 mlfl of Adekanol LG 294 (trade name). The Philippine molasses was diluted with water so that the saccharide content for the cell propagation tank might be adjusted to 150 g/l and the saccharides content for the alcohol fermentation tank might be 400 g/l.
The fermentation mediums were sterilized at 120C for 10 min and were fed to the respective fermentation tanks.
As a flocculating microorganism, Saccharomyses cerevisiae IR~2 was used, and the ethanol fermentation was carried out according to Fig. 2. This flocculating microorganism was preliminarily grown in a usual manner .. , before being introduced into the present process.
Through the line 4, the molasses medium having a saccharides content of 150 g/l was introduced into the cell propagation tank 1 at such a flow rate that the dilution rate was 0.227 h-l. At that time, 10~ of the liquid in the cell propagation tank was inoculated. The supply of oxygen was carried out by passing air into the cell propagation tank; the gas flow rate, the oxygen concentration, and the carbon dioxide concentration were measured by the respiratory-quotient (RQ)-measuring apparatus 7, to find the RQ, and the amount of the passed air and the number of stirring rotations were controlled so that the value of RQ might be 22 to 27. The operation was carried out with the amount of the passed air in the range of 0.3 to 0.5 VVM. The stirring blades were of the propeller type and the number of rotations was in the range of 200 to 250 rpm. The fermentation temperature was 30C and the pH was kept at 4.75 to 4.8.

At that time, in the cell propagation tank 1, the cell concentration became 90 to 100 g~l in terms of the dry weight, the alcohol concentration became 64 to 65 g/l, and the remaining saccharide concentration became 0.2 g/l or less. This ~ermentation liquid was introduced into the settling separation tank 3 and all of the liquid flowed out therefrom was introduced into the fermentation .
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20~860 tank 2. At that time the cell concentration of the liquid flowed out was 10 g/l. All of the separated cells were returned to the cell propagation tank 1.
On the other hand, the molasses medium having a 5 saccharides concentration of 400 g/l was supplied to the fermentation tank 2 with the dilution rate ~eing 0.033 h-l, and as a supply of oxygen, air was used with a feeding speed of 0.1 VVM. The stirring blades were of the propeller type and the number of rotations was 100 rpm.
The fermentation temperature was 30C and the pH was kept at 4.75 to 4.8.
At that time, in the fermentation tank 2, the cell concentration could be kept at 70 to 90 g/l and the alcohol concentration could be kept at as high as 87 to 90 g/l. The remaining saccharide concentration became 3 g/l or less.
' The dilution rate to the whole fermentation tank volume in this test became 0.108 h-1. Under these operating conditions, the operation was possible stably and continuously for 1,000 hours or more.
Comparative ExamPle 2 After Example 2, an alcohol fermentation test was performed using only one tank, which was the fermentation tank 2. The fermentation medium was the same molasses medium that was used in Example 1. The .
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, 20~4860 saccharide concentration was 180 g/l and the dilution rate was the same as that to the whole fermentation tank volume of Example 1. Other fermentation tank conditions were the same as those in Example 2.
As a result, the cell concentration in the fermentation tank 2 gradually decreased to 30 g/l and the alcohol concentration also decreased, so that stable operation was impossible.
When fermentation was performed similarly using only one tank with the cell concentration being 400 g/l and the dilution rate being 0.05 h-l, the remaining saccharides concentration became 200 g/l or more and settling and separation became difficult, so that stable operation was impossible.
ComParative ExamPle 3 Example 2 was repeated, except that RQ was kept at 1.'8. Since the flocculating activity of the cells was low, the cell concentrations of the cell propagation tank of the first step and the alcohol fermentation tank of the second step did not increase, so that stable operation was impossible.
Further, when the operation was performed with the operating conditions being changed so that RQ might be kept at 300l the cell concentration of the cell propagation tank 1 was restored, but since the amount of 20S~860 produced cells was small and the fermentation activity was low, the remaining cell concentration of the alcohol fermentation tank 2 became 150 g/l or more, so that stable operation was impossible.
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Having described our invention as related to the present embodiments, it is our intention that the invention not be limited by any of the details of the description, unless otherwise specified, but rather be construed broadly within its spirit and scope as set out in the accompanying claims.

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Claims (22)

1. A process for continuously fermenting saccharides to alcohol using a flocculating microorganism as an alcohol fermentation microorganism, which comprises effecting the production of alcohol in at least two steps;
in the first step, a stirring-type cell propagation tank equipped with an oxygen supply apparatus and a cell separation apparatus is used, so that flocculating microorganisms substantially high in fermentation activity are continuously grown; and said flocculating microorganisms are fed to a fermentation tank of the second step for continuously producing highly concentrated alcohol.

2. The process as claimed in claim 1, wherein oxygen is fed into the cell propagation tank of the first step in a feeding rate of 1 to 1,500 mo1/m3?hr.

3. The process as claimed in claim 1, wherein the temperature in the cell propagation tank of the first step is controlled in the range suitable for the cell propagation, and the temperature in the fermentation tank of the second step is controlled in the range suitable for the alcohol fermentation.

4. The process as claimed in claim 1, wherein the cell concentrations in the first and second step are kept at 10 g/l or more, respectively.

5. The process as claimed in claim 1, wherein the concentrations of saccharides to be fed and the feeding rates of saccharides in the first step and the second step are each enable to change, independently.

6. The process as claimed in claim 1, wherein the concentration of saccharides to be fed to the cell propagation tank is 1 to 20 wt.%.

7. The process as claimed in claim 1, wherein the concentration of saccharides to be fed to the fermentation tank is 20 wt.% or more.

8. The process as claimed in claim 1, wherein each remaining saccharide concentration in the first step and the second step is 50 g/l or less.

9. The process as claimed in claim 1, wherein the remaining saccharide concentration in the fermentation tank of the second step is 10 g/l or less.

10. The process as claimed in claim 1, wherein the alcohol concentration of the cell propagation tank of the first step is 70 g/l or less.

11. The process as claimed in claim 1, wherein the alcohol concentration of the fermentation tank of the second step is 70 g/l or more.

12. A process for continuously fermenting saccharides to alcohol using a flocculating microorganism as an alcohol fermentation microorganism, which comprises at least two steps; wherein in the first step, a stirring-type cell propagation tank equipped with a respiratory quotient-measuring apparatus, an oxygen supply apparatus, a temperature controller, and a cell separation apparatus is used; the quantity of propagation of cells and the fermentation yield of alcohol are controlled using the respiratory quotient (RQ) as an index, so that flocculating microorganisms substantially high in fermentation activity are continuously grown, and in the second step, said flocculating microorganisms grown are fed to a fermentation tank for continuously producing highly concentrated alcohol equipped with an oxygen supply apparatus, a temperature controller, and a cell separation apparatus.

13. The process as claimed in claim 12, wherein the respiratory quotient at the cell propagation tank in the first step is controlled in the range from 2 to 200.

14. The process as claimed in claim 12, wherein the temperature in the ceil propagation tank of the first step is controlled in the range suitable for the cell propagation, and the temperature in the fermentation tank of the second step is in the range suitable for the alcohol fermentation.

15. The process as claimed in claim 12, wherein the cell concentrations in the first and second step are kept at 10 g/l or more, respectively.

16. The process as claimed in claim 12, wherein the concentrations of saccharides to be fed and the feeding speeds of saccharides in the first step and the second step are each enable to change, independently.

17. The process as claimed in claim 12, wherein the concentration of saccharides to be fed to the cell propagation tank is 1 to 20 wt.%.

18. The process as claimed in claim 12, wherein the concentration of saccharides to be fed to the fermentation tank is 20 wt.% or more.

19. The process as claimed in claim 12, wherein each remaining saccharide concentration in the first step and in the second step is 50 g/l or less.

20. The process as claimed in claim 12, wherein the remaining saccharide concentration in the fermentation tank of the second step is 10 g/l or less.

21. The process as claimed in claim 12, wherein the alcohol concentration of the cell propagation tank of the first step is 70 g/l or less.

22. The process as claimed in claim 12, wherein the alcohol concentration of the fermentation tank of the second step is 70 g/l or more.