GB2104914A - Process for manufacturing alcohol by fermentation - Google Patents

Abstract

In a process for manufacturing alcohol by fermentation employing immobilized microbial cells, a single fermenter (1) is used for both immobilization of microbial cells by gellation and the subsequent fermentation. Preferably any flocs which precipitate during the fermentation process are removed at (4) to prevent contamination of the immobilized microorganism. Also the yield of alcohol can be increased by subjecting culture liquor recovered from the fermenter to further fermentation in a second fermentation vessel. <IMAGE>

Description

SPECIFICATION Process for manufacturing alcohol by fermentation This invention relates to a process for manufacturing alcohol by fermentation employing immobilized microorganisms.

The production of ethanol by fermentation processes using a microorganism immobilised on a carrier, is known, but despite a long carrier life the conventional processes for manufacturing alcohol by fermentation employing immobilized microorganisms are unsatisfactory because of eventual contamination of the microorganism by various germs which lower its activity. It is therefore necessary to prevent contamination of the microorganism by various germs by sterilizing the apparatus and the medium, by maintaining a positive pressure throughout the apparatus, 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, the growth of the immobilized microbial cells is inhibited, 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. Linko and P. Linko: Biotechnology Letters, 3(1), 21 to 26 (1981)1. 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 cells, resulting in difficulty in continuation of stable fermentation.

The present invention seeks to provide a process for the manufacture of alcohol wherein stabilized fermentation can be continued for a long period of time without inactivation of the immobilized microorganisms due to contamination by germs, such as acetic acid producing bacteria, which converts ethanol to acetic acid. In accordance with the present invention it has been found that the high activity of immobilized ethanol-producing fermentation microorganisms can be maintained over long periods of time by performing the immobilization of the microorganism and the fermentation in a single vessel.

It has also been found, in accordance with this invention, that the contamination of the immobilized microbial cells by various germs is largely due to the flocculation of the germs with the alcohol-producing microorganism and which together form fine flocs which settle in the fermenter and are difficult to remove therefrom.

Accordingly, in a preferred aspect of the present invention the fermentation process is carried out with periodic or continuous removal of the flocs which form in the fermentation medium during the fermentation process. In this way the high activity of the microorganisms is effectively preserved and the fermentation process can be continued over a long period of time.

In accordance with yet another aspect of this invention it has been found that a culture liquor overflowing a fermenter always contains 108 to 108 living cells/ml, which is a number substantially equal to that of the microbial cells employed in conventional batch fermentation, and that they have an ability to produce alcohol.

Therefore, it is possible in accordance with the present invention to further extend the fermentation process by continuing the fermentation in a second fermenter using culture liquor from the first fermenter.

The invention will be further described hereinafter with reference to the accompanying drawings, in which: Figure 1 shows a fermenter having a means for settling and removing flocs in accordance with the present invention 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, but modified in accordance with the present invention to permit the removal of flocs from the culture medium.

Numbers in Figures 1-3 denote the following.

1: Fermenter, 2: Membrane of immobilized microorganisms, 3, 17: Inclined plate, 4, 10, 14: Outlet of culture liquor, 5, 15: Outlet of gas, 6, 13: Precipitate collecting area, 7, 8, 18: Inlet of medium, 9, 12: Granular immobilized microbial cells, 11, 16: Wire net.

Referring now to the present invention in more detail, the microorganism is first immobilized in situ in the fermentation vessel. This may be done in a conventional manner for example by gelling a mixture of living microbial cells and an aqueous solution of an immobilizing agent such as a monovalent alkali metal salt of alginic or pectic acid and a derivative thereof, or sodium salt of carrageenan by adding thereto in a fermentation vessel an aqueous solution of a polyvalent metal salt or potassium salt. After formation of the gel containing the immobilized microbial cells, excess gelling agent is removed and the vessel charged with the culture medium ready for commencement of the fermentation process. Suitable examples of the derivatives of pectic acid include its partial 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 Al+++. Therefore, they are suitably used in the preparation of the immobilized microbial cells employed for the process of this invention.

The immobilization of living microbial cells is achieved by a known method, 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.

A carrier is placed or secured in a fermenter and an immobilizing agent solution containing microbial 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 discharged after the immobilization.

The immobilization of the microbial cells must be performed under completely sterile conditions.

After the immobilization, an appropriate culture medium is supplied 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 wtiv % of immobilizing agent solution containing 103 to 108 microbial cells/ml. The immobilization is carried out at a temperature of 20 to 350C and at a pH suitable for activating the microbial cells.

According to this invention, it is also possible to obtain an improved yield of alcohol from sugar by incubating in the second fermenter a culture liquor overflowing 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 alcohol in a high concentration.

According to this invention, it is preferable to maintain a sugar concentration of 1 5 to 20% at the inlet of the first fermenter, and 2 to 5% at the outlet 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 concentration of 8 to 1 1% are usually maintained in the second fermenter. As the concentration of sugar left in the second fermenter becomes lower, the yield of alcohol from sugar becomes higher and the retention time becomes longer. The retention time is usually determined so that the quantity of residual sugar in the second fermenter can be maintained within the range hereinbefore mentioned.

According to this process for continuous fermentation, 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 immobilizing agent which permits liberation of the microorganisms to a certain extent. 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 second 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.

Descriptions will now be made of some embodiments of the apparatus used according to the process of this invention for effecting alcoholic fermentation, while collecting flocs effectively.

Figure 1 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 microorganisms are immobilized. Flocs of germs and the microorganism are collected in a precipitative collecting area 6, and discharged through an outlet 4.

Figure 3 shows a fermenter comprising a conical precipitation vessel having an inclined plate 1 7. 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 the effluent containing alcoholic product is continuously and quantitatively removed with pump, etc.

For comparison purposes, Figure 2 shows a conventional 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.

According 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 900C to 1 000C for about five minutes, depending on the microorganisms to be used, and the influence of various germs thereon. According to the process of this invention, however, sterilization can be carried out at a temperature as low as 700C without involving any possibility of contamination by various germs. The process of this invention is of great significance, since a lot of energy has hitherto been required for sterilization of the material to prevent contamination by various germs.

The invention will now be described more specifically with reference to several examples thereof.

Example 1 As an alcohol-producing microorganism, wine yeast No. 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 CaCI2 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 CaCI2 solution is removed. Then, molasses containing 15 w/v % of 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 1 5 days of culturing. The concentration of alcohol shows a gradual reduction from 8% to 6% on the 30th day. The value of 6% corresponds to a yield of 63% on the basis of the quantity of sugar, when the theoretical value is considered as 100%.

Example 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 I-fermenter with the arrangement shown in Figure 1. After sterilization, the fermenter is charged with a mixed solution of sodium alginate and the seed culture prepared 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 supplied to the fermenter from the bottom, whereby a gel in the shape of a film is formed on each of the sheets of cotton cloth. Then, a culture medium consisting of molasses containing 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 l/HR to 3.5 I/HR, and ethanol production can be steadily continued at a rate of 65 to 68 g/l 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 concentration of alcohol is 50 g/l on the 15th day, and 40 g/l on the 30th day.

Example 3 Two columns of the type shown in Figure 2, each having a capacity of three I 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 1 of the gel is left in each column, and the other gel is discharged. An aqueous solution of blackstrap molasses having a sugar content of 1 50 g/l is continuously supplied in a rising flow into one of the columns (column No. 1) 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 300C.

In both of the columns, alcohol production increases with the growth of the immobilized cells, and becomes constant on the fifth day, when ethanol shows a concentration of 60 g/l, and residual sugar a concentration of 1 6 g/l at the outlet of column No. 1. Ethanol shows a concentration of 56 g/l, and residual sugar a concentration of 41 g/l at the outlet of column No.

2. The activity of the immobilized microbial cells in column No. 1 lowers on the sixth day of constant production, and on the 10th day, the concentration of alcohol decreases to 46 g/l with a corresponding increase in the concentration of residual sugar to 60 g/l. In column No. 2, however, there is no lowering of the activity of the immobilized microbial cells after about two months of continuous fermentation, and the production of alcohol can be continued satisfactorily.

A part of the culture liquor obtained from column No. 2 and having an alcohol content of 56 g/l is supplied via a closed conduct to a second 2.2 I fermenter continuously 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/l of ethanol.

Example 4 The same procedures as in Example 3 are repeated except that the carbon dioxide produced in the first fermenter (column No. 1) is supplied to the second fermenter. Ethanol is steadily obtained from the second fermenter at a concentration of 69 g/l.

Example 5 A precipitation vessel is provided at the outlet of the second 1 I fermenter in Example 4. The culture liquor from the second fermenter is caused to overflow the top of the precipitation vessel, and the precipitated microbial cells are returned to the second fermenter. The concentration of microorganism in the second fermenter increases to 10 g/l (dry), and ethanol is steadily obtained at a concentration of 72 gIl.

Example 6 Four kinds of immobilized microbial cells are prepared by employing two kinds of natural high molecular substances, i.e., sodium alginate and pectate, and two kinds of synthetic materials, i.e., 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 1 50 g/l is used as a culture medium.

The quantity of the living microorganisms is measured at the outlet of each column in which ethanol is steadily produced at a concentration of 45 to 50 gjl. It is 5x 107 cells/ml in the sodium alginate column, 4x107 cells/ml in the sodium pectate column, 6x 108 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 temperature of 300C. Ethanol is produced from the effluent of the sodium alginate column at a concentration of 13 g/l, and from the cellulose acetate butyrate column at a concentration of 5 girl.

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 girl, and further containing 0.5 g/l ammonium sulfate, 0.2 g/l potassium hydrogenphosphate, and'0.l g/l yeast extract. The culture medium is supplied into the column at a rate of 150 ml/HR, and when the reaction becomes steady, ethanol is produced at a concentration of 88 g/l. On the 10th day of steady state, lowering of the activity of the immobilized microbial cells is observed, and the concentration of ethanol decreases to 60 g/l on the 20th day.When the culture 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/l. The culture liquor obtained by supplying the culture medium at a rate of 450 ml/HR is supplied into 2.2 1 second fermenter at a rate of 100 ml/HR. The fermenter is maintained at a temperature of 300C with stirring. An alcohol concentration of 86 to 90 g/l was steadily obtained at the outlet of the second fermenter 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 No. 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 Figure 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 1 5% of sugar is heated at 900C 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 I). Then, a molasses solution sterilized by heating at 1 200C for 1 5 minutes is supplied for continuous fermentation.

The feed is gradually increased to obtain a sugar concentration of 20 g/l 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 each fermenter at a feed rate of 800 ml/HR. The fermentation is continued, and the contamination by various germs is examined. The sugar is steadily converted to alcohol in a yield of 95% (relative to the theoretical value of 100%) without any growth of various germs for 500 hours. After 500 hours of culturing, the molasses solution is sterilized for Experiments 2 and 3 at 900C for five minutes as for Experiment 1. After 800 hours of culturing, a reduction in yield to 80% is observed in Experiment 2, and flocs of various germs are found in the column.In Experiments 1 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 70at, and the fermentation is continued. After 1,200 hours of fermentation, Experiment 1 shows a yield of 91%, Experiment 2 shows a yield of 61%, and Experiment 3 shows a yield of 85%. In Experiment 1, 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, 1 I of aqueous solution containing 10% sodium salt of tc-carrageenan (Soageena MV-101, Mitsubishi Acetate Co.) and 0.5% locust bean gum (Soalocust A-200, Mitsubishi Acetate Co.) in 2.5 1 first fermenter of the type shown in Figure 2 is sterilized and 0.05 ml of seed culture of Example 1 is added thereto.

The mixture is added dropwise to 2 l of 2% potassium chloride solution in the first fermenter to form beads of gel. After incubation for one hour, molasses solution containing 1 6 w/v % of sugar is added at a rate of 0.8 l/HR and air is fed thereto at a rate of 0.1 1/min. After 6 days of continuous fermentation, molasses solution is fed at a rate of 0.8 l/HR to obtain culture liquor containing 9.5 v/v % alcohol.

Then 50 1 second fermenter is fed with the culture liquor from the first fermenter at a rate of 0.8 I/HR and fresh molasses solution containing 60 wt/v % of sugar at a rate of 0.2 l/HR for 48 hours. The culture liquor is incubated for 48 hours to obtain ethanol in a concentration of 1 5 v/v %.

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.

Claims (5)

Claims

1. A fermentation process for the production of alcohol which comprises culturing an immobilized alcohol-producing microorganism in a fermentation medium and recovering the alcohol produced, wherein the immobilization of the microorganism and the fermentation process are carried out in one and the same vessel.

2. A process according to claim 1, wherein the immobilized microorganism is formed in situ in the fermentation vessel by initially charging to the fermentation vessel a mixture comprising the microorganism and an aqueous solution of an alkali metal salt of alginic or pectic acid and adding thereto a salt of polyvalent metal thereby to form a gel comprising the microorganism.

3. A process according to claim 1, wherein the immobilized microorganism is formed in situ by the gellation of an aqueous solution comprising the microorganism the sodium salt of carrageenan with a polyvalent metal slat or potassium salt as the gelling agent.

4. A process according to claim 1,2 or 3 wherein any flocs which form as precipitates in the fermentation medium are continuously or periodically removed from the fermentation vessel.

5. A process according to claim 1, 2, 3 or 4, wherein the fermentation process is carried out on a continuous basis, and culture liquor recovered from the fermentation vessel is subjected to further fermentation in a second fermentation vessel.