JPS6013675B2 - Production method of high concentration ethanol using immobilized bacteria - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to bacteria of the genus Zymomonas (hereinafter simply referred to as immobilized bacteria) having ethanol-producing ability immobilized on a gel-like carrier.

), and in more detail, it is a method for producing high-concentration ethanol that involves contacting and reacting immobilized bacteria with a medium containing nutrients necessary for growth within the gel and fermentable sugars that serve as raw materials for ethanol production. This is a method for producing concentrated ethanol. Since ancient times, ethanol has been produced by fermentation using yeast.

However, its production is carried out in batch operations, and although a culture solution containing 50 to 70 females' ethanol can be obtained in 20 to 4 field hours of fermentation, it is difficult to produce ethanol in high concentrations. requires fermentation time of over a month. Moreover, it requires a great deal of labor and expense to recover and reuse the yeast after fermentation has been completed in a batch operation, which is not an economically advantageous method. In recent years, methods have been developed to produce useful substances using immobilized microorganisms. For example, yeast is immobilized on calcium alginate gel and the complex enzyme system of Sonada Ikyou yeast is used to produce ethanol from glucose. It has also been reported that this can be done. (Bio ch.BiMng 1
However, the ethanol production capacity of fixed I○ yeast is low, with a maximum of 50 females/kite producing ethanol in a long reaction, and its productivity is low. It is low in nutrients and does not contain the nutrients necessary for the growth of yeast cells.
Since the contact reaction is carried out with a solution containing only glucose as an ethanol raw material and calcium chloride for gel retention, there is a problem in that the activity of the enzyme rapidly decreases as the reaction time progresses.

Previously, the present inventors completed a new method for preparing immobilized microorganisms that makes effective use of the complex enzyme system of microorganisms by allowing a dense microbial cell layer to remain in a gel carrier (particularly Kao No. 53-41471). The present inventors have demonstrated that the immobilized bacteria obtained by immobilizing Zymomonas bacteria capable of producing ethanol using this method for preparing immobilized microorganisms exhibits an ethanol productivity of approximately 1. (For example, ethanol with a concentration of about 50 9' can be produced in a reaction of about 45 minutes.)
.

Furthermore, when the immobilized bacteria are supplied with a nutrient tower containing about 9 parts of 100 fermentable sugars and nutrients necessary for bacterial cell proliferation, the concentration of fermentable sugars decreases as the reaction progresses. However, if a nutrient tower containing a high concentration of fermentable sugars is additionally supplied when the concentration drops to below, for example, 10 females/secretion, the immobilized bacteria will be able to increase their productivity without decreasing their performance. The performance of the immobilized bacteria can be improved by expressing the ability to efficiently produce concentrated ethanol, and by adding a fresh medium containing nutrients necessary for bacterial cell growth to the immobilized bacteria and performing the reaction in the same machine. can be retained for a long period of time,
Therefore, by repeating these operations, approximately 200
It has been found that it is possible to stably produce ethanol with a high concentration of up to 9/100 m. That is, according to the present invention, Zymomonas bacteria immobilized on a gel-like carrier and capable of producing ethanol are brought into contact with a medium containing fermentable sugars of about 50 to 100% and nutrients necessary for the growth of the bacteria. When the fermentable sugar concentration in the reaction solution has decreased to about 20% or less of the initial concentration, a fresh nutrient medium containing more than about 100 ml of sugar is added continuously or intermittently. By replenishing 3, ethanol can be produced at a high concentration.

In the present invention, as Zymomonas bacteria having ethanol-producing ability, Zymomonas bacteria having strong ethanol-producing ability, such as Zymomonasis mopilis IF○
13756, Zymomonas anaerogia ATCC2
9501 etc. can be suitably used.

The immobilization of Zymomonas bacteria capable of producing ethanol is usually carried out by the microbial cell immobilization method (e.g. carrageenan gel method, polyacrylamide gel method, etc.) described in the literature.
``In particular, Samurai Isoaki 53
It is preferable to carry out the preparation according to the method for preparing immobilized microorganisms described in the specification of No. 141471.

The immobilized bacteria according to the method of the same patent application contains in advance viable bacterial cells equivalent to 0.0 to 10 platinum loops per 100 units of gel (green weight), and has a granular or A gel that can be formed into a membrane is prepared by a known gel entrapment method, and then the gel is incubated at 25q0 to 40°C in a nutrient medium suitable for the growth of the bacteria to retain a high concentration of viable bacterial cells within the gel. It is prepared by instillation. Here, as the gel base, for example, agar, carrageenan, tuff-cerelan, sodium alginate, polyvinyl alcohol, cellulose succinate, casein succinate, 2-methyl-5-pyridine, methyl acrylate/methacrylic acid copolymer, etc. can be used. can. 0 The production of high concentration ethanol of the present invention can be carried out either by a batch method or by a continuous method using a column.

In the batch method, first an organic nitrogen source, such as yeast extract, cornstap liquor, beptoso, yeast echitas, a vitamin source, ammonium salts, phosphates, organic acids, magnesium salts, calcium salts, sodium salts, potassium salts. Prepare a medium containing the nutrients necessary for bacteria to grow in the gel, and add to it about 10% of the medium, i.e. about 9/100, of non-fermentable sugars such as glucose and molasses. and prepare a nutrient medium containing fermentable sugars.

When the above-mentioned immobilized bacteria are added to this medium and subjected to a contact reaction while being stirred, fermentable sugars are quickly assimilated to produce ethanol, and at the same time, the sugar concentration in the reaction solution decreases. If the sugar concentration is, for example, about 20% or less of the initial concentration,
Preferably, if the fermentable sugar is replenished when the concentration is less than about 9/I, the fermentable sugar will be assimilated in the same manner as above and ethanol will be produced, and at the same time, the sugar concentration in the reaction solution will decrease again. come. If the fermentable sugar is replenished when the sugar concentration in the reaction solution decreases like this, the maximum
It is possible to produce ethanol as high as ethanol. The fermentable sugar additionally supplied to the reaction solution in the above is preferably a nutrient medium containing fermentable sugar at a high concentration of 100 9' or more, and the sugar concentration in the soil of the replenishment tower is increased each time it is replenished. increase continuously or intermittently, approximately 100 to 40
All you have to do is make it about 9/desk of 0. In addition, when using a continuous method, a column packed with immobilized bacteria is used, and a nutrient medium containing fermentable sugars of about 9/100 is initially supplied continuously from one end of the column, and the immobilized bacteria produce ethanol in the column. is produced, and the reaction-completed liquid is discharged from the end of the column.

In this case, the feeding rate of the culture medium is adjusted so that the concentration of fermentable sugar in the effluent is about 20% or less of the initial Z-phase concentration, preferably about 10% or less, and By increasing the fermentable sugar concentration in the supplied medium, the immobilized bacteria will develop the ability to efficiently produce ethanol over a long period of time, allowing ethanol to be stabilized for a long period of time at a high Z degree of up to 200 c'. can be produced. Furthermore, by connecting several columns in series or by using a long column, the concentration of fermentable sugar in the middle of the column or in the middle of each column is 20% or less of the initial concentration, preferably about 10 mo'. By adjusting the supply rate of the culture medium so that the sugar concentration decreases and supplying the fermentable sugar-containing nutrient tower to the areas where the sugar concentration decreases, it is possible to increase the production capacity of high-concentration ethanol per unit time. Ethanol can be produced more efficiently and continuously. 2. In this specification, all sugar concentrations are expressed as values converted to glucose.

Furthermore, in the following examples, the ethanol production ability of immobilized bacteria was determined from the amount of ethanol produced. Example
1 3 Zymomonas
One platinum loop of Mobilis IFO 13756 was added to 20 skins of an aqueous carrageenan solution sterilized at 3700 and mixed.

This mixed solution was added dropwise to 200 g of a 2% potassium chloride aqueous solution from a nozzle to form a spherical gel with a diameter of 4 sides.
3 This gel-immobilized bacteria was mixed with yeast extract 0.15%, ammonium chloride 0.25%, dipotassium hydrogen phosphate 0.55%, magnesium sulfate, heptahydrate 0.
．． 025%, calcium chloride 0.001%, citric acid 0
．． 1% and 0.25% sodium chloride, pH 6
．． Glucose was added to the nutrient medium (hereinafter simply referred to as Toji) adjusted to a concentration of 10% to give a concentration of 10%. Bacterial cells were allowed to grow within the gel with static pupils. The immobilized bacteria thus obtained were 77 9 ethanol/
Gel/hr ethanol production was expressed. Next, these 20 immobilized bacteria were placed in a culture medium containing 10% glucose and allowed to stand still at 3'0, and when the remaining amount of glucose in the reaction solution became 2 females/secretion after 4 powders, the glucose 4
The reaction was continued under the same conditions by replenishing 0%-containing column material No. 10'.

As a result, the performance of immobilized bacteria did not deteriorate, and the total reaction time was 2
．． After 0 hours, 100 women's high-concentration ethanol was added to the
[was able to produce. Example 2 20' of the immobilized bacteria prepared as in Example 1 were grown in a medium containing 10% glucose at 30°C.
The reaction mixture was allowed to react for 40 minutes, and the glucose concentration in the reaction solution was adjusted to 2M'.

Next, this reaction solution was replenished with medium 5 containing 40% glucose, and thereafter this replenishment was repeated 3 times every 40 minutes under the same conditions.
I returned the line. As a result, the immobilized bacteria maintained an ethanol production chamber of 77 9 ethanol/gel/h and yielded an ethanol solution of 125 g/40' after a total reaction time of 3 hours and 20 minutes.

Example 3 After performing the same operations as in Example 2, the immobilized bacteria were collected, and ethanol production was repeated in the same manner as in Example 2.

Even after repeating the reaction 10 times, the ethanol production ability of the immobilized bacteria did not decrease, and 40 ethanol solutions of 125 o' were obtained every 3 hours and 20 minutes of reaction time. Example 4 Immobilized bacteria 20' obtained in the same manner as in Example 1 were placed in a cylindrical column (capacity 30
First, three cylindrical columns were prepared containing immobilized yeast, which had been treated by feeding the yeast containing 10% glucose at a rate of 3000 hr for 9 hours. are connected in series, and No. 1 is connected upward from the bottom column. 1.No. 2.No. 3 (see Figure 1 for the equipment).

No. A medium containing 10% glucose was added to the bottom of column No. 1 at a rate of 30 secretions/hr. The No. 2 column has NO. The effluent from 1 was supplied as is, and a medium containing 40% glucose was also secreted for 7'h.
It is fed together with the effluent at a rate of r. No. No. 3 column has NO. The effluent from 2 was fed as is, and the medium containing 40% glucose was also fed at a rate of 7 [/hr]. Thus no. From column No. 3, a reaction solution containing ethanol at a concentration of 100 9' was continuously obtained at a rate of 44 hours. Example 5 In Example 2, a 20% molasses aqueous solution (glucose equivalent concentration: 100
Using [) in 9', the reaction was carried out in the same manner as in Example 2, increasing the bran syrup concentration to 60% in a gradient manner, thereby producing an ethanol solution of 125/9/40 in a total reaction time of 3 hours. got '.

Example 6 Immobilized bacteria 25 obtained in the same manner as in Example 1 were transferred to a cylindrical column equipped with a reflux tube for circulating a part of the reaction solution from the middle of the column to the bottom of the column (the apparatus is shown in Figure 2). ), and at 30qC, add 20% bran syrup aqueous solution (see ) instead of glucose at 30qC.
Glucose equivalent concentration: 100 9' secretion) was supplied from the bottom end of the column at a rate of 40 hours for 9 hours to mobilize the immobilized bacteria, and the reaction-completed solution was allowed to flow out from the top end at the same rate.

Next, 50% molasses aqueous solution (glucose equivalent concentration: 250
9/') was fed at a rate of over 15/hr, and part of the reaction solution in the column was circulated at a rate of 100/hr. The concentration of molasses in terms of glucose in the circulating fluid is 10
It dropped to below 9/gu. Since the molasses in the supply medium was diluted by this circulation operation, the ethanol preforming ability of the immobilized bacteria in the column did not decrease, and an ethanol-containing effluent of 125 9/ship could be continuously produced.

[Brief explanation of the drawing]

FIG. 1 shows a schematic diagram of each ethanol production apparatus when a cylindrical column is used, and FIG. 2 shows a schematic diagram of each ethanol production apparatus when a cylindrical column having a reflux tube is used. In Figures 1 and 2, 1, 2, and 3 are columns that can be filled with immobilized bacteria, and 4, 5, and 6 are columns containing highly concentrated fermentable sugars*
Reference numeral 7 indicates a supply port for the nutrient medium, an outlet for the reaction-completed solution, and 8 a reflux tube for circulating the reaction solution. Little brother 2 illustration

Claims (1)

[Scope of Claims] 1 Zymomonas bacteria having ethanol-producing ability immobilized on a gel-like carrier are mixed with about 50 to 100 mg of fermentable sugar/
ml and a culture medium containing nutrients necessary for bacterial growth, and when the fermentable sugar concentration in the reaction solution has decreased to about 20% or less of the initial concentration, about 100 mg/m
1. A method for producing high-concentration ethanol, which comprises continuously or intermittently supplementing a fresh nutrient medium containing 1 or more sugars. 2. The method according to claim 1, wherein the culture medium is brought into contact with the immobilized Zymomonas bacteria while continuously or intermittently increasing the concentration of fermentable sugar in the medium to be replenished. 3. When carrying out a contact reaction between the immobilized Zymomonas bacteria and the medium by continuously supplying the medium to a column filled with the immobilized Zymomonas bacteria, the fermentable sugar concentration in the reaction completed solution is 20% or less of the initial concentration. 3. The manufacturing method according to claim 1 or 2, wherein the replenishment rate of the medium is adjusted so that the concentration of the medium is low.