Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
I'ITLE: PROCES~S~ ~OL BY FER~ ENTA~['ION
BACKGF~OUND OF THE INVENTION
1. Field of the Invention:
, This invention relates to a process for manufac-
turing alcohol by fermentation employing immobilized micro-
organisms~
2. Description of the Prior Art:
lQ The conventional process for manufacturing alcohol
by fermentation employing immobilized microorganisms is
unsatisfactory because of contamination by various germs,
despite a long carrier life. It i5 necessary to prevent
contamina-tion by various germs by sterilizing the apparatus
and the medium, by nlaintaining a positive pressure throughout
the apparatus r and by protecting the immobilized microbial
cells against contamination during -transfer from one vessel
to another.
In the continuous alcoholic fermentation employing
immobilized microbial cells, as the quantity of ethanol
increases with a decrease in the quantity of residual sugar r
the growth of the i~lobilized microbial cells is inhibited r
and their activity is lowered~ It is known that alcohol
can be produced continuously at a yield of 60 to 80% for a
long time [Y. Lin~o and P. Linko: Biotechnology L2tters r
3 (1) r 21 to 26 (1981)]. Production of ethanol in a yield
of more than 80% brings about a lowering of the physiological
activity of the immobilized microorganisms, and death of a
part of the microbial cellsr resulting in difficulty in
continuation of stable fermentation.
SU~ARY OF THE INVENTION
An ob~ect of this invention is to provide a
process for the manufacture of alcohol wherein stabilized
fermentation is continued for a long period of time by
protecting the immobilized microorganisms against contamination
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by germs such as acetic acid-producing bacteria which con
verts ethanol to acetic acid. It has been found that the
high activity of the immobilized microorganisms can be
maintained for a long time by performing the immobilization
of the microorganisms and the continuous fermentation in a
single vessel.
It has also been found that the contamination of
the immobilized microbial cells by various germs is due to
the flocculation of the germs with the alcohol-producing
microorganism forming fine flocs which settle in the fer-
menter and are difficult to remove therefrom. It is, there-
fore, another object of this invention to provide a process
for the manufacture of alcohol wherein the immobilized
microorganisms can be protected against contamination by
various germs by removing the flocs of the germs and micro-
organisms effectively. This ob~ect is attained by providing
the vessel with a means for collecting the flocs.
A further object of this invention is to provide
a process for manufacturing alcohol at a high yield by con-
tinuous fermentation for a long time. It has been foundthat a culture liquor overflowing a fermenter always con-
tains 106 to 108 living cells/ml, which is a numbex sub-
stantially equal to that of the microbial cells employed
in conventional batch fermentation, and that they have an
ability to produce alcohol. This object is, therefore,
attained by producing alcohoi in a first fermenter in a
concentration which does not inhibit the ability to produce
alcohol of the immobilized microbial cells, and continuing
the fermentation in a second fermenter.
BRIEF DESCRIPTION OF THE DRAWINGS
.
F IGURE 1 shows a fermenter having a means for
settling flocs, and a membrane of immobilized microorganisms;
FIGURE 2 shows a conventional fermenter employing
granular immobilized microorganisms; and
FIGURE 3 shows a fermenter employing granular
immobilized microorganisms.
Numbers in Figuxes 1- 3 denote the following.
1: Fermenterl 2: Membrane of immobilized microorganisms,
3, 17: Inclined plate~ 4, 10, 14: Outlet of culture liquor,
5, 15: Outlet of gas, 5, 13: Precipitate collecting area,
7, 8, 18: Inlet of medium, 9, 12: Granular immobilized
microbial cells, 11, 16: Wire net.
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DETAILED DESCRIPTION OF THE INVENTION
According to the present invention, fermentation
is continued for a long time by gelling in a fermenter a
mixture of liviny microbial cells and an aqueous solution
of an immobilizing agent such as a monovaIent alkali metal
salt of alginic or pectic acid and a derivative thereof, or
sodium salt of carrageenan with an aqueous solution of a
polyvalent metal salt or potassium salt as a gelling agent
to immobilize the microbial cells, removing excess gelling
agent and adding a culture medium for fermentation. Suitable
examples of the derivatives of pectic acid include its par~
tial alkyl ester.
Although an aqueous solution of a monovalent alkali
metal salt of alginic or pectic acid, or a derivative thereof
does not form a gel even at a low temperature, it is easily
gelled by contact with a solution of a salt of a polyvalent
metal ion such as Ca or Al . Further, carrageenan is
gelled by contact with a solution of a metal ion such as K ,
Ca or A1 . Therefore, they are suitably used in the
preparation of the immobilized microbial cells employed for
the process of this invention.
The immobili~ation of living microbial cells is
achieved by a known m~thod, such as the formation of a
granular gel, or immobilization on the surface of any of
various types of carriers. An immobilizing agent solution
containing microbial cells is added dropwise to a solution
of a gelling agent in a fermenter to form a granular gel~
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A carrier is placed or secured in a fermenter and
an immobilizing agent solution containing microbia] cells
is added thereto to adhere the cells to the carrier.
A solution of a gelling agent is added thereto to obtain
the microbial cells immobilized on the carrier. In either
method, excess solution of the immobilizing agent is dis-
charged after the immobilization.
The immobilization of the microbial cells must be
performed under completely sterile conditions. After the
immobilization, an appropriate culture medium i5 supplled
to carry out fermentation.
The immobilization of the microbial cells can be
easily accomplished according to known methods. That is,
0.5 to 10% of gelling agent solution is added to 0.5 to 30
wt/v ~ of immobilizing agent solution containing 103 to 109
microbial cells/ml. The immobilization is carried out at
a temperature of 20 to 35C and at a pH suitable for acti-
vating the microbial cells.
According to this invention~ it is also possible
to cbtain an improved yield of alcohol from sugar by
incubating in the second fermenter a culture liquor over-
flowing the first fermenter until a high concentration of
alcohol is obtained. The second fermenter is preferably of
the complete mixing type. Since a yeast is an agglutinative
microorganism, it is effective to perform agitation for
obtaining an improved reaction rate. For agitation in the
second fermenter, it is preferable to use the carbon dioxide
produced in the first fermenter. There is no substantial
contamination by germs in the second fermenter, since the
concentration of alcohol is high.
The contamination can be almost prevented when
the concentration of alcohol is more than 10%.
The second fermenter is connected to the first
fermenter with an appropriate pipe, etc.
The whole apparatus is made sterile in advance.
If required fresh medium is added to the second fermenter
to obtain alcchol in a high concentration.
1~9~
~ ccordiny to this invention, it is preferable to
maintain a sugar concentration of 15 to 20% at the inlet of
the first fermenter, and 2 to 5% at the cutlet of the first
fermenter, and an alcohol concentration of 6.5 to 9% at the
outlet. A sugar concentration of 1.5 to 2%, and an alcohol
concentra-tion of ~ to 11~ are usually maintained in the
second fermenter. As the concentration of sugar le~t in
the second fermenter becomes lowex, the yield of alcohol
from sugar becomes higher and the retention time becomes
longer. rrhe re-tention time is usually determined so that
the quantity of residual sugar in the second fermenter can
be maintained within the range hereinbefore mentloned.
According to this process for continuous fermen-
tation, as the concentration of free microorganisms in the
culture liquor overflowing the first fermenter is higher,
the time required for fermentation in the second fermenter
is shorter. Accordingly, it is preferable to employ immobi-
lizing agent which permits liheration of the microorganisms
to a certain e~tent. The use of a natural high molecular
substance, such as alginic acid,pectic acid or carrageenan
is preferred, since it permits liberation of nearly 10 times
as large a quantity of microorganisms from the immobilized
microbial cells as that from any synthetic carrier.
A calcium alginate gel is particularly preferred, since it
is tough and easy to use and is superior in cell liberation.
According to this invention, the microbial cells
flowing out of the second fermenter can be collected by,
for example, a centrifugation, and returned to the second
fermenter to raise the concentration of the cells therein
to shorten the retention time. By evacuating the secon~
fermenter or introducing a large quantity of gas thereto,
a part of alcohol can be removed from the liquid to reduce
any adverse effect caused by alcohol in the second fermenter
to achieve improved productivity.
If required, it is possible to employ a plurality
of second fermenters positioned in parallel or additional
fermenters arranged in series to one another~
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Descriptions will now be made of some embodiments
of the apparatus used according to the process of this inven-
tion for effecting alcoholic fermentation, while collecting
flocs effectively.
FIGU~ l shows a fermenter 1 provided at its
bottom with an inclined plate 3 having a lower end at which
a suspended precipitative substance is collected, and through
which the effluent is discharged. The fermenter employs a
plurality of membranes on which alcohol-producing microorgan-
isms are immobilized. Flocs of germs and the microorganismare collected in a precipitative collecting area 6, and dis-
charged through an outlet 4.
FIGURE 3 shows a fermenter comprising a conical
precipitation vessel having an inclined plate 17. This
fermenter employs granular immobilized microbial cells in
the form of beads 12. Flocs are collected in a precipitative
collecting area 13 through a wire net 16, and discharged
through an outlet 14.
In both fermenters, culture medium is introduced
through an inlet in the upper part of the fermenter and
culture liquor is discharged through an outlet in the lower
part of the fermenter with flocs.
The culture medium is continuously introduced into
these fermenters and ~he effluent containing alcoholic pro-
duct is continuously and quantitatively removed with pump,
etc.
For comparison purposes, FIGURE 2 shows a conven-
tional fermenter system having an inlet 8 and an outlet 10
in which granular immobilized microbial cells 9 are employed.
In these three fermenters, flocs can be directly
taken out of system.
~ccording to the process of this invention, there
is virtually no contamination by various germs, even if the
material to be fermented is sterilized at a temperature
lower than that usually employed. It has hitherto been
usual to sterilize the material at a temperature of ~0C
to 100C for about five minutes, depending on the micro-
organisms to be used, and the influence of various germs
3~3~
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thereon. According to the process of ! his invention,
however, sterili~ation can be carried out at a temperature as
low as 70C without involving any possibility of contamina-
tion by various germs. The process of this invention is of
S great significance, since a lot of energy has hitherto been
required for sterilization o~ the material to prevent con-
tamination by various germs.
The invention will now be described more specifi
cally with reference to several examples thereof.
EXAMPLE 1
_
As an alcohol-producing microorganism, wine yeast
~2 of the Brewage Association is used. 30 ml of the seed
culture liquor is mixed carefully with 300 ml of a 3% aqueous
solution of sodium alginate. The mixture is added dropwise
through a nozzle to a fermenter containing one liter of a
sterilized 2% aqueous solution of CaC12 to form gels in bead
form as shown in Figure 2. After the gel is left to stand
for one hour to age the immobilized cells, the excess CaC12
solution is removed. Then, molasses containing 15 w/v %
o~ sugar is supplied at a rate of 300 ml/HR continuously to
grow a large quantity of yeasts in the gel. From the fourth
day of culturing, molasses is fed at a rate of 300 ml/HR,
and alcohol production at a yield of 8.8 v/v % is continued
for about three months without any lowering of the activity
of the immobilized cells.
For comparison, immobilization and fermentation
are conducted separately. The growth of various germs is
observed after about 15 days of culturing. The concentra-
tion of alcohol shows a gradual reduction from 8% to 6% onthe 30th day. The value of 6% corresponds to a yield of
63% on the basis of the quantity of sugar, when the theore-
tical value is considered as 100%.
FXAMPLE 2
_ _ _
A carrier is prepared by juxtaposing several
sheets of cotton cloth of 20 cm by 30 cm at intervals of
3 mm, and placed in a 7 ~-fermenter with the arrangement
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sho~n in Figure 1. After sterilizatlon, the fermenter is
charged with a mixed solution of sodium alginate and the
seed culture preparéd in the same manner as in Example 1.
After the excess solution is removed from the bottom of the
fermenter, a sterilized 2~ solution of calcium chloride is
supplled to the fermenter from the bottom, whereby a gel in
the shape of a film is forrned on each of the sheets of cotton
cloth. Then, a culture medium consisting of molasses con-
tainlng 15~ of sugar is supplied to the fermenter and the
culture liquor is recovered from the outlet of the fermenter.
Carbon dioxide is exhausted from the top of the fermenter.
The flow rate of the culture medium is gradually increased from
1 Q/~R to 3.5 Q/~R, and ethanol production can be steadily
continued at a rate of 65 to 68 g/Q for a month without
causing any trouble, such as blocking of the fermenter
(Experiment 1~.
For comparison, the carrier immersed in the mixed
solution is removed from the fermenter, and immobilization
is performed with a 2% solution of calcium chloride in a
fermenter in a sterile box. A carrier supporting a film of
immobilized cells is transferred into the fermenter.
Molasses is supplied in the same manner as in Experiment 1
above. Flocs are formed by the growth of various germs in
the bottom of the fermenter, and gradually increase. The
concentxation of alcohol is 50 g/Q on the 15th day, and
40 g/Q on the 30th day.
EXA LE 3
Two columns of the type shown in Figure 2, each
having a capacity of three Q are filled with a 2~ aqueous
solution of calcium chloride. A mixed solution containing
nine parts of a sterilized 3.3~ aqueous solution of sodium
alginate and one part of a wine seed culture liquor of
Example 1 is dropped through a nozzle into each column to
form spherical particles of a gel having a diameter of about
4 mm. About 1.5 Q of the gel is left in each column, and
the other gel is discharged. An aqueous solution of black-
strap molasses having a sugar content of 150 g~Q is contin-
g
uously supplied in a rising flow into one of the columns(column No. l) at a rate of 450 ml/HR, and into the other
column (column No. 2~ at a rate of 1,200 ml/HR. The column
temperature is maintained at 30C. In both of the columns,
alcohol production increases with the growth of the immobi-
lized cells, and becomes constant on the fifth day, when
ethanol shows a concentration of 60 g/Q, and residual sugar
a concentration of 16 g/Q at the outlet of column No~ l.
Ethanol shows a concentration of 56 g/Q, and residual sugar
a concentration of 41 g/Q at the outlet of column No. 2.
The activity of the immobilized microbial cells in column
No~ l lowers on the sixth day of constant production, and
on the 10th day, the concentration of alcohol decreases to
46 g/Q with a corresponding increase in the concentration
of residual sugar to 60 g/Q. In column No. 2, however,
there is no lowering of the activity of the immobilized
microbial cells after about two months of contlnuous fermen-
tation, and the production of alcohol can be continued satis-
factorily.
A part of the culture liquor obtained from column
No. 2 and having an alcohol content of 56 g/Q is supplied
via a closed conduct to a second 2.2 Q fermenter contin-
uously at a rate of 200 ml/HR. The volume of the culture
liquor in the fermenter is kept to be two liters. The second
fermenter steadily contains 62 g/Q of ethanol.
EXAMPLE 4
The same procedures as in Example 3 are repeated
except that the carbon dioxide produced in the irst fer-
menter (column No. l) is supplied to the second fermenter.
Ethanol is steadily obtained from the second fermenter at a
concentration of 69 g/Q.
EX~MPLE 5
A precipitation vessel is provided at the outlet
of the second 1 Q fermenter in Example 4. The culture liquor
from the second fermenter is caused to overflow the top of
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the precipitation vessel, and the precîpitated microbialcells are returned to the second fermenterO The concentra
tion of microorganism in the second fermenter increases to
10 g/Q (dry~, and ethanol is steadily obtained at a concen-
tration of 72 g/Q~
EXAMPLE 6
Four kinds of immobilized microbial cells are
prepared by employing two kinds of natural high molecular
subs-tances, i.e., sodium alginate and pectate, and two kinds
of synthetic materials, i.eO, cellulose acetate butyrate
and porous polystyrene. 50 ml of each of the immobilized
cells is charged into a 100 ml column of the type shown in
Figure 2. A molasses solution having a sugar content of
150 g/Q is used as a culture medium. The quantity of the
living microorganisms is measured at the outlet of each
column in which e-thanol is steadily produced at a concen-
tration of 45 to 50 g/Q. It is S x 10 cells/ml in the
sodium alginate column, 4 x 107 cells/ml in the sodium
pectate column, 6 x 106 cells/ml in the cellulose acetate
butyrate column, and 3 x 106 cells/ml in the polystyrene
column The effluent of each of the sodium alginate and
cellulose acetate butyrate columns is supplied at a rate of
20 ml/HR into a 200 ml second fermenter kept at a tempera-
ture of 30C. Ethanol is produced from the effluent of thesodium alginate column at a concentration of 13 g/Q, and
from the cellulose acetate butyrate column at a concentra-
tion of 5 g~Q.
EXAMPLE 7
The procedures of Example 3 are repeated except
for the use of a culture medium consisting of a molasses
solution having a sugar content of 200 g/Q/ and further
containing 0.5 g/Q ammonium sulfate, 0.2 g/Q potassium hydro-
genphosphate, and 0.1 g/Q yeast extract. The culture mediumis supplied into the column at a rate of 150 ml/HR, and
when the reaction ~ecomes steady, ethanol is produced at a
3~
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concentration of 88 g~Q. On the 10th day of steady state,
loweriny of the activity of the immobilized microbial cells
is observed, and the concen-tration of ethanol decreases to
60 g/Q on the 20th dayO When the cul-ture medium is supplied
at a rate of 450 ml/HR, the activity of the immobilized
microbial cells is stable for more than two months, though
the concentration of ethanol is 65 g/Q. The culture liquor
obtalned by supplying the culture medium at a rate of 450
ml/HR is supplied into 2.2 Q second fermenter at a rate of
100 ml/HR. The fermenter is maintalned at a tempera-ture of
30C with stirring. An alcohol concentration of 86 to 90
g/Q was steadily obtained at the outlet of the second fer-
menter for a long time.
EXAMPLE 8
.
Each of the apparatus shown in FIGURES 1 to 3 is
sterilized. Two volumes of a culture liquor containing wine
yeast #2 of the Brewage Association are admixed with one
volume of a sterilized 10% aqueous solution of sodium
alginate. The mixture is supplied to the fermenter shown
in FIGIJRE 1, whereby the membrane is impregnated with the
mixture, and a 2'~ aqueous solution of calcium chloride is
supplied to form a film of gel. Then, a molasses solution
containing 15% of sugar is heated at 90C for five minutes
and is supplied for continuous fermentation (Experiment 1).
The sterilized calcium chloride solution is also supplied to
each of the apparatus shown in FIGURE 2 (Experiment 2~ and
FIGURE 3 (Experiment 3). The mixture containing sodium
alginate and yeasts is dropped into each fermenter to form
beads of gel (1.5 Q). Then, a molasses solution sterilized
by heating at 120C for 15 minutes is supplied for continuous
fermentation.
The feed is gradually increased to obtain a sugar
concentration of 20 g/Q at the outlet of each of the vessels
shown in FIGURES 1 to 3. After four days, a culture liquor
having an alcohol concentration of 8.2 to 8.4 v/v ~ is
obtained from sach fermenter at a feed rate of 800 ml/HR.
The fermentation is continued, and the contamination by
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various ge~ms is examined. The sugar is steadily converted
to alcohol in a yield of 95% (rela-tive to the theoretical
value of 100%) wi-thout any growth of various germs for 500
hours. After 500 hours of culturing, the molasses solution
is sterilizedfor Experiments ? and 3 at 90C for five minutes
as for Experiment l. After 800 hours of culturing, a reduc
tion in yield to 80% is observed in Experiment 2, and flocs
of various germs are found in the column. In Experiments l
and 3, a yield of 95% is maintained, and no floc of various
germs is found. Then, the temperature for the sterilization
of the molasses solution is lowered to 70C, and the fermen-
tation is continued. After 1,200 hours o~ fermentation,
Experiment l shows a yield of 91%, Experiment 2 shows a
yield of 61%, and Experiment 3 shows a yield cf 85%.
In Experiment l, the yield is 90% even after 1,800 hours of
fermentation, whereas 60% yield in Experiment 2 and 82%
yield in Experiment 3.
EXAMPLE 9
-
In this example, l Q of aqueo .15 solution containing
10% sodium salt of K-carrageenan (Soageena MV-101, Mitsubishi
Acetate Co.) and 0.5% locust bean gum (Soalocust A-200,
Mitsubishi Acetate Co.) in 2~5 Q first ermenter of the type
shown in Figure 2 is sterilized and 0.05 ml of seed culture
of Example l is added thereto. The mixture is added drop-
wise to 2 Q of 2% potassium chloride solution in the first
fermenter to form beads of gel. After incubation for one
hour, molasses solution containing 16 w/v % of suyar is
added at a rate of 0.8 Q/HR and air is fed thereto at a rate
of 0.1 Q/min. After 6 days of continuous fermentation,
molasses solution is fed at a rate of 0O8 Q/HR to obtain
culture liquor containing 9~5 v/v ~ alcohol.
Then 50 Q second fermenter is fed with the culture
liquor from the first fermenter at a rate of 0.8 Q/HR and
fresh molasses solution containing 60 wt/v % of sugar at a
rate of 0.2 Q/HR for 48 hours. The culture liquor is incu-
bated for 48 hours to obtain ethanol in a concentration of
15 v/v %.
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The fermentation of the second fermenter is
repeated using several second fermenters to obtain culture
liquor containing alcohol in a high concentration for three
months.
