JPH053780A - Method for separating microorganism - Google Patents

Abstract

PURPOSE:To form firm aggregates of microbial cells excellent in settling properties and efficiently separate the microbial cells by regulating an aqueous suspension of microorganisms to an acidic state and then adding sodium polyacrylate to the regulated aqueous suspension. CONSTITUTION:An aqueous suspension of microorganisms is regulated to pH <=4 and sodium polyacrylate in an amount of preferably >=0.002 based on the dry weight of the suspended microbial cells is then added to form aggregates of the microbial cells. The resultant aggregates are then separated by a method for normally settling the aforementioned aggregates and separating liquids, etc. The pH is normally regulated by using an acid such as sulfuric acid or hydrochloric acid.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for efficiently separating microbial cells from a suspension of microorganisms, particularly bacteria.

[0002]

2. Description of the Related Art As a method for producing a useful substance by using a microorganism, a useful substance is accumulated inside the microbial cell, and it is released outside the microbial cell and accumulated in water in which the microorganism is suspended. There is a way. It is necessary to collect the microbial cells when the useful substance is accumulated in the microbial cells, and conversely, it is necessary to remove the microbial cell when the useful substance is accumulated outside the microbial cells. Become. In any of these methods, it is necessary to separate microorganisms in a suspended state in order to isolate and purify useful substances.

As a method for separating microorganisms, a method using centrifugation is known. When a Sharpless centrifuge is used, the effect of separating the bacterial cells and the supernatant liquid is excellent, but the bowl needs to be replaced frequently depending on the amount of microorganisms, and there is a problem in operability. This drawback can be remedied by using a continuous centrifuge, but due to the structure of the device, strong centrifugal force cannot be applied, and complete separation of microorganisms is difficult.

Since the size of microbial cells is as small as 0.5 to 2.0 μ, it cannot be filtered using a filter paper or filter cloth.
Therefore, a method is known in which the microbial suspension is adjusted to pH 4 and heated and hot filtered together with activated carbon that serves as a filter aid (JP-A-59-28488). In this method, when the useful substance is accumulated in the aqueous phase outside the cells, depending on the type of the useful substance, the useful substance is adsorbed on the activated carbon to cause a large loss. On the other hand, when useful substances are accumulated in the microbial cells, the microbial cells cannot be collected because it is difficult to recover them.

The method of forming aggregates of microbial cells and allowing them to settle by gravity is a method of improving these drawbacks, and several methods have been proposed. For example, a method of adding polyaluminum chloride to adjust the pH to 5 to 8 and then adding sodium polyacrylate (JP-A-49-1)
33585), a method of adding a cationic polymer flocculant (JP-A-61-40784), a method of adding a chloride (JP-A-63-27648), a method of adding boric acid or a salt thereof to adjust the pH to 8 or more. (JP-A-2-26547
0), a method of adding calcium hydroxide to form an aggregate of microbial cells at a pH of 10 or more, and subsequently adding ferric chloride to grow and separate the aggregate (Japanese Patent Publication No. 55-49).
826) and the like are known. These methods cause denaturation depending on the type of useful substance, and have problems with aggregating property and sedimentation property, so that cell separation is not satisfactory.

[0006]

As a method for separating microbial cells, a method using centrifugation or filtration has the above-mentioned problems, and a method that is satisfactory as a method for forming aggregates of microbial cells is also available. unknown. The problem to be solved by the present invention is to more effectively form aggregates of microorganisms and separate the microbial cells than conventionally known.

[0007]

[Means for Solving the Problems] As a result of intensive investigations by the present inventors, a method for separating microbial cells by forming an aggregate of microbial cells suspended in water If the suspension of cells is adjusted to pH 4 or lower and sodium polyacrylate is added, the sedimentation is good,
It was found that a strong aggregate of microbial cells was formed, and the present invention was completed based on this finding.

That is, according to the present invention, an aqueous suspension of microorganisms is
It is adjusted to H4 or less and sodium polyacrylate is added to form aggregates of bacterial cells to separate microorganisms.

For example, when the microorganism is Escherichia coli, as shown in Reference Example 1, a small amount of sodium polyacrylate is added to a bacterial cell suspension having a pH of 4 or less to form a bacterial cell aggregate, and Sedimentation occurs. However, even if an anionic polymer flocculant of acrylamide-acrylic acid copolymer is added, a lump of microbial cells is not formed.
Sodium polyacrylate in an acidic solution and acrylamide-acrylic acid copolymer are presumed to cause a difference in the dissociation of the carboxyl group and the shape of the polymer molecule, resulting in the formation of aggregates of microorganisms. It

An acid may be added to adjust the pH of the microbial suspension to 4 or less, but the kind of the acid is not limited, and inorganic acids such as sulfuric acid, hydrochloric acid, phosphoric acid and nitric acid, as well as formic acid, acetic acid, Organic acids such as citric acid can be used.

Sodium polyacrylate is available in the form of powder in the market. The powder may be added as it is to the microbial suspension, or an aqueous solution may be prepared and added to the microbial suspension. After sodium polyacrylate is dispersed and dissolved in the entire microbial suspension, an aggregate of bacterial cells is immediately formed. As shown in Reference Example 2, microbial cell aggregates are formed only by adding a trace amount of sodium polyacrylate to the microbial cell suspension having a pH of 4 or less. However, in order to form the aggregates most effectively, It is desirable to add 0.002 times or more of sodium polyacrylate based on the dry weight of the suspended microbial cells. However, even if a large excess of sodium polyacrylate is added, it does not lead to an increase in the agglomeration effect in proportion to the added amount, and thus sodium polyacrylate is wasted.

The microbial suspension in the method of the present invention means a suspension in which microorganisms (for example, bacteria, yeast, mold, actinomycetes, single cell algae, etc.) are suspended. As shown in Reference Example 2, the microorganism may be in a suspended state in a culture solution in which various kinds of salts and organic substances are mixed, or it may be washed thoroughly,
It may be suspended in water, physiological saline, buffer solution or the like. That is, in order to form aggregates of microbial cells, it is sufficient to adjust the cell suspension to pH 4 or less and then add sodium polyacrylate, without requiring the coexistence of specific components.

Further, after synthesizing a useful substance by using as a catalyst the microbial cells themselves having a high ability to produce a specific enzyme, the reaction solution is adjusted to pH 4 or less, and sodium polyacrylate is added to the lumps of microbial cell residues. It is also possible to recover the aqueous phase in which the useful substance is dissolved by forming For example, it can be applied to the step of removing microorganisms when purifying amino acids produced using the microorganisms. To give a few examples, Escherichia coli
a coli), Coryneb
genus Lactobacillus, Brevibacterium (Brev
When L-tryptophan is produced by utilizing the physiological actions of the bacterium of the genus ibacterium, the physiological actions of the bacteria of the genus Escherichia coli, the genus Citrobacterium, the genus Erwinia are used. For example, the case of producing 3,4-dihydroxy-L-phenylalanine (hereinafter abbreviated as L-DOPA) can be exemplified.

The temperature for forming aggregates of microbial cells is not limited. However, at high temperature, the liquid convection causes the aggregates to float, making it impossible to settle. When the lumps of microbial cells are settled and separated to separate the microbial cells, it is desirable to carry out in a temperature range in which the flocculated lumps do not float depending on the characteristics of the container in which the microbial cells are aggregated. ..

Since the aggregate of bacterial cells thus formed is larger than that of normal bacterial cells, the sedimentation property is improved. Therefore, in order to separate the bacterial cells from the aqueous phase, let stand to allow the aggregate of bacterial cells to settle to the bottom of the container,
A method of separating the liquid can be adopted. In addition, since normal cells lack centrifugal force, good separation results can be obtained even with a continuous centrifuge, which has a problem. On the other hand, since a high sterilization effect can be obtained by using a smaller amount of filter aid than that used in the case of filtering normal bacterial cells, when useful substances are accumulated in the water solvent, bacterial cells by filtration are Separation becomes easy. Activated carbon is generally used as a filter aid, but when the useful substance is a substance that is easily adsorbed by activated carbon, the loss is greatly reduced.

[0016]

EXAMPLES The present invention will be specifically described below with reference to Reference Examples, Examples and Comparative Examples. Reference Example 1 The enzyme tryptophan synthase (EC 4.2.1.2
0) Escherichia coli having activity (Esc.
herichia coli) MT-10242 (FE
The RM BP-20) strain was inoculated on the LB agar plate medium of Table 1 and cultured overnight at 35 ° C.
A 500 ml Sakaguchi flask equipped with a cotton plug containing 150 ml of LB liquid medium shown in Table 2 was inoculated. The inoculated Sakaguchi flask was shake-cultured at 35 ° C for 24 hours (120 rp).
m) 20 L of this broth was added to the PT minimal medium of Table
Transfer to the prepared 30l jar fermenter and transfer to 35
Culturing was carried out at 0 ° C., an aeration rate of 1 vvm, and a stirring rotation speed of 600 rpm. At this time, the pH of the culture solution was adjusted to pH 6.8.
Ammonia water was added by a controller for control, and sterilized 40% glucose aqueous solution was sequentially added, and culture was performed for 40 hours.

90 ml culture solution (E. coli concentration 29 g / l)
Was placed in a 100 ml graduated cylinder (inner diameter 2.7 cm), adjusted to various pHs in the range of pH 2 to pH 9 with sulfuric acid or sodium hydroxide, and kept at 25 ° C. 0.5%
After adding 1 g of an aqueous solution of sodium polyacrylate, water was added to bring the total amount to 100 ml and mixed well. While keeping the temperature at 25 ° C. as it was, it was allowed to stand for 30 minutes, and the volume of the precipitated bacterial cell phase was measured. The result was as shown in FIG. When the pH of the culture solution in which Escherichia coli was suspended was 4 or less, aggregates of bacterial cells were formed and settled at the bottom of the measuring cylinder, but at higher pH, aggregates of bacterial cells were not formed and sedimentation did not occur. I couldn't see it.

[0018]

[Table 1]

[0019]

[Table 2]

[0020]

[Table 3]

Reference Example 2 The cells suspended in the culture solution cultured by the method of Reference Example 1 were collected by centrifugation, suspended in water and centrifuged again to wash. 11.1 g / l, 5.0 g / l, 2.2 g /
1 E. coli aqueous suspension (adjusted to pH 4 with sulfuric acid) was prepared. On the other hand, the culture medium cultivated by the method of Example 1 was p
Adjusted to H4. 90 ml of each of these E. coli suspensions was placed in a 100 ml graduated cylinder (inner diameter 2.7 cm) and kept at 25 ° C. The amount of 0.5% sodium polyacrylate aqueous solution added was varied to obtain various sodium polyacrylate concentrations, and water was added to bring the total amount to 100 ml and mixed well. While keeping the temperature at 25 ° C. as it was, the mixture was allowed to stand for 30 minutes, and the volume of the precipitated bacterial cell phase was measured. The result was as shown in FIG. The amount of sodium polyacrylate to be added is 0.002 times or more the dry weight of the suspended cells. In addition, since the same aggregates were formed in the washed bacterial cells, it can be seen that it is not necessary to allow a specific substance to coexist. The supernatant liquid after the sedimentation of the bacterial cells was clear.

Example 1 Escherichia col
i) MT-10242 (FERM BP-20), Corynebacterium glutamicum (Corinebact)
erium glutamicum) (ATCC 21
253), Brevibacterium flavum (Brev
ibacterium flavum) (ATCC 2
1128), Bacillus subtilis
S. subtilis) (ATCC 14618) was inoculated into the LB agar medium of Table 1 and cultured at 35 ° C. overnight. Two 500 ml Sakaguchi flasks with a cotton wire containing 150 ml of the LB liquid medium shown in Table 2 were inoculated with 2 platinum loops of the cells grown on the LB agar medium. The inoculated Sakaguchi flask was shake-cultured (120 rp for 24 hours at 35 ° C).
m) After culturing, mix the culture solutions in the two flasks,
95 ml was dispensed into two 100 ml measuring cylinders. The pH was adjusted to 4.0 with sulfuric acid, 1 g of 0.5% sodium polyacrylate aqueous solution was added to one graduated cylinder to a constant volume of 100 ml, and the other graduated cylinder was added with 100 ml without adding anything. , And mixed well. The mixture was allowed to stand for 30 minutes while keeping the temperature at 25 ° C., and the volume of the bacterial cell phase was measured. The results shown in Table 4 were obtained. In all the bacteria, the addition of sodium polyacrylate improved the sedimentation of the bacteria.

[0023]

[Table 4]

Example 2 3.4 in terms of dry weight of the cells obtained by the method of Reference Example 1.
g, L-serine 25 g, pyridoxal phosphoric acid 0.02
After being dissolved in water together with g to make the total amount 206 g, an aqueous sodium hydroxide solution was added to adjust the pH to 8.3. A solution prepared by dissolving 25 g of indole in 89 g of toluene was added to this aqueous solution. When stirred at 35 ° C. for 20 hours, 41.9 g of L-tryptophan was accumulated. The pH of this solution was adjusted to 4 with sulfuric acid, and water was added to bring the total volume to 900.
It was diluted to g and heated to distill off the toluene. Water was added to dilute the total amount to 1000 g, and the mixture was heated to 80 ° C. to obtain a solution of L-tryptophan.

500 g of this L-tryptophan solution
While maintaining the temperature (containing 21.0 g of L-tryptophan) at 80 ° C., add 5% of 0.5% sodium polyacrylate aqueous solution.
g, and gently stirred for 15 minutes to form an aggregate of bacterial cells. Transfer this to a sufficiently heated centrifuge tube and
Centrifugation was carried out for 5 minutes at 0 G of centrifugal force. Sufficiently sterilized to obtain 490 g of a clear supernatant liquid, which contained 20.1 g of L-tryptophan. The recovery rate of L-tryptophan after the disinfection operation was 95.2%.

Comparative Example 1 500 g of L-tryptophan solution prepared in Example 1
Without adding sodium polyacrylate to (containing 21.0 g of L-tryptophan), it was placed in a sufficiently heated centrifuge tube and centrifuged at a centrifugal force of 3500 G for 5 minutes. However, the supernatant was not clear because the cells could not be separated from the liquid phase. From the comparison with Example 1, it can be seen that centrifugation is facilitated by forming an aggregate of bacterial cells.

Example 3 Enzyme tyrosine phenol lyase (EC 4.1.9)
9.2) Escherichia coli having activity, Escherichia coli (E
scherichia coli) MT-10473
The strain (FERM P-10182) was designated as LB-A in Table 5.
P agar plate medium was inoculated and cultured overnight at 30 ° C., and 2 platinum loops each of the grown bacterial cells were placed in 4 500 ml Sakaguchi flasks with 100 ml of LB-AP liquid medium in Table 6 with a cotton plug. I inoculated. Inoculated Sakaguchi flask is 30 ℃
Shake culture (120 rpm) for 12 hours, and after culturing for a predetermined time, the culture medium of Escherichia coli having tyrosine phenol lyase activity shown in Table 7 was transferred to a 30-liter jar fermenter charged with 20-liter, and the aeration rate was 1 vvm at 30 ° C. The culture was carried out at a stirring rotation speed of 600 rpm. At this time, ammonia water was added and controlled by a pH controller so that the culture solution had a pH of 6.8, and the culture was continued for 38 hours while successively adding a sterilized 40% glucose aqueous solution.

9.0 g of the cells thus obtained, in terms of dry weight, was converted into 20 g of L-serine and 20 of catechol.
g, boric acid 12.4 g, 2-mercaptoethanol 0.
5 g, EDTA 0.5 g, pyridoxal phosphoric acid 0.05
After being dissolved in water together with g to make the total amount 500 g, the pH was adjusted to 9.0 with aqueous ammonia. After stirring at 30 ° C. for 20 hours while purging with nitrogen, L-DOPA
68.8 g was accumulated. Add sulfuric acid to this solution to adjust the pH.
Adjust to 4, dilute the total amount to 2000 g with water, add 2 g of hydrosulfite, heat to 80 ° C. and add L-
A DOPA solution was obtained.

500 g of this L-DOPA solution (L-DOPA
DOPA (containing 28.6 g) was kept at 80 ° C. and kept at 0.
5 g of a 5% sodium polyacrylate aqueous solution was added and gently stirred for 15 minutes to form an aggregate of bacterial cells. This was allowed to stand for 30 minutes to allow the aggregate of bacterial cells to settle, and the supernatant was pumped up to collect 455 g of an L-DOPA aqueous solution. When 50 g of warm water at 80 ° C. was added to 50 g of the aggregated phase of the settled cells, the mixture was gently stirred and allowed to stand,
The aggregate of bacterial cells settled again. Pump up the supernatant liquid phase, 50
g of L-DOPA aqueous solution was recovered. L-DOPA was mixed with the supernatant obtained after the first settling of bacterial cell aggregates and sedimentation.
505 g of an aqueous solution containing 27.3 g was obtained. This L-DO
Since the bacterial cell aggregate was slightly mixed in the PA aqueous solution, 1.0 g of powdered activated carbon was added, the mixture was stirred at 80 ° C. for 30 minutes, hot filtered, and the activated carbon was washed with 100 g of hot water. .. Thus, 500 g of an L-DOPA aqueous solution containing no aggregate of bacterial cells was obtained. L-DOPA was contained in an amount of 26.8 g, and the recovery rate was 93.7%.

[0030]

[Table 5]

[0031]

[Table 6]

[0032]

[Table 7]

Comparative Example 2 500 g of L-DOPA solution obtained by the method of Example 2
5.4 g of powdered activated carbon in (containing 28.6 g of L-DOPA)
Was added, and the mixture was stirred at 80 ° C. for 30 minutes, hot-filtered, and the activated carbon was washed with 65 g of hot water. Thus, 523 g of an aqueous L-DOPA solution containing no cells was obtained. L-DOPA
Was contained in an amount of 25.5 g, and the recovery rate was 89.3%.

If the amount of activated carbon as a filter aid is less than this amount, the rate of hot filtration will be drastically reduced and it will be impractical. At the same time, the amount of L-DOPA adsorbed on the activated carbon was large, and L-DOP was added to the sterilized solution as compared with the case of Example 3.
The recovery rate of A is low. This example also shows the effectiveness of the method for removing bacterial cells by forming aggregates of bacterial cells using sodium polyacrylate.

[Brief description of drawings]

FIG. 1 is a diagram showing a pH range in which an aggregate of Escherichia coli is formed by the addition of sodium polyacrylate.

FIG. 2 is a diagram showing the amount of sodium polyacrylate added necessary to form E. coli aggregates.

Claims (1)

Claims: 1. An aqueous suspension of microorganisms is adjusted to pH 4 or less, and sodium polyacrylate is added to the suspension.
A method for separating a microorganism, which comprises forming an aggregate of bacterial cells.