JPH0731488A - Separation of bio-polyester from bio-polyestercontaining microorganism - Google Patents

Abstract

PURPOSE:To provide a method for efficiently separating a bio-polyester in a granular state from microbial cells containing the bio-polyester. CONSTITUTION:This method for separating the granular bio-polyester comprises charging the aqueous suspension of bio-polyester-containing microorganisms into a pressure-resistant container or preliminarily heating the suspension at 40-100C and then charging the heated suspension into the pressure-resistant container, and subsequently heating and retaining the charged suspension at 40-100C for raising the pressure to spout the suspension from the small opening of the container, thus allowing the shearing force of the fluid to act on the microorganisms.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for separating biodegradable biopolyester from bacterial cells.

[0002]

2. Description of the Related Art Currently, plastic wastes are treated by incineration, landfill, etc., but these treatment methods have problems such as global warming and land relaxation of landfill sites. For this reason, as the social awareness of plastic recycling increases, a recycling system is being developed. However, recyclable uses are limited, and as a practical matter, many plastic waste treatment methods cannot be handled by incineration, landfill, and recycling alone, and many are left in the natural environment. Therefore,
Biodegradable plastics that are taken into the natural material cycle after disposal and whose decomposition products do not become harmful substances are drawing attention, and their development is underway. As such a plastic, in particular, polyester produced by microorganisms in the cells is expected to be incorporated into the natural carbon cycle process to stabilize the ecosystem. Also,
In the medical field as well, it can be used as an implant material or a drug carrier that does not require recovery.

However, in order to use this polyester as a plastic, it is necessary to separate it from the microbial cells and take it out. As a method for obtaining a biopolyester from a biopolyester-containing microorganism, an extraction method with an organic solvent such as chloroform, hypochlorite soda (Williamson, DH, and Wil)
kinson, J .; F. (1958), J. Gen. M
icrobiol. 19, 198-203. ) Or lysozyme is used to dissolve bacterial cells and the remaining polymer is recovered as granules. In addition, a method of recovering a polymer by dissolving bacterial cells with a specific enzyme other than lysozyme (JP-A-60-145097), 10
A method in which cells are destroyed by releasing the pressure of steam or the like having a high pressure of more than 0 ° C. and separated into cell debris and a polymer (Japanese Patent Laid-Open No. Sho 5).
7-174094) and the like.

[0004]

However, the solvent extraction method using chloroform or the like requires a large amount of not only the extraction solvent but also a poor solvent for reprecipitation. Therefore, if each solvent is to be reused, it is necessary to separate the two solvents. Furthermore, since it is generally necessary to completely dry the entire microbial cell prior to solvent extraction, a large amount of heat energy is also required. Therefore, in order to industrially produce biopolyester, many process equipments are required. And energy is required, which is a practical disadvantage. When treated with hypochlorous soda, the drawbacks of the solvent extraction method can be avoided, but on the other hand, the molecular weight of the polyester decreases (JA Ramsay, E. Berger,
B. A. Ramsay and C.I. Chavaric
(1990), J. Am. Biotechnology Te
chniques 4, 4, 221-226), which causes problems with the quality of the polymer. Enzymes like lysozyme
It is effective for a small amount of experimental use, but it is not suitable for mass production of biopolyester because it is difficult to secure a large amount. In the enzyme method disclosed in JP-A-60-145097, the operations before and after the enzyme treatment have multiple stages, and there is still a lot of room for improvement for mass production. The effect of the method of releasing pressure of JP-A-57-174094 is unclear because the purity and yield of the obtained polyester are not described. An object of the present invention is to provide a method for separating a biopolyester from a microorganism containing a biopolyester by applying a shearing force to a microbial cell in an aqueous medium of less than 100 ° C without using an organic solvent.

[0005]

The present invention introduces an aqueous suspension of biopolyester-containing microorganisms into a pressure resistant container,
Alternatively, the suspension is preheated to a temperature range of 40 to 100 ° C. and introduced into a pressure-resistant container, heated and kept in the temperature range of 40 to 100 ° C., a high pressure is applied to the suspension, and The present invention relates to a method for separating bio-polyester from a bio-polyester-containing microorganism, which comprises applying a fluid shearing force to the micro-organism by ejecting the suspension from the opening to separate granular bio-polyester. Incidentally, in the method of JP-A-57-174094, the high pressure is also used, but this destroys the microbial cells by the pressure shock when the high pressure is suddenly reduced, whereas the present invention uses the high pressure liquid. This is a method of promoting cell destruction and separation of biopolyester by the shearing force at the time of jetting with a fine mouth.

The microorganism used in the present invention is a bacterium (cell) accumulating biopolyester in the cell. For example, Alcaligenes
s), A. lipolytica AK201 (Japanese Patent Laid-Open No. 5-64592), A.L. eutrophus, A .;
Latus, etc., Pseudomonas (Pseudomon)
As), Bacillus genus, Azotobacterium genus, Nocardia genus, and the like strains are shown, but the strains are not limited thereto.

Here, biopolyester means poly-
It refers to a microorganism-produced polyester called polyhydroxyalkanoate [hereinafter abbreviated as P (HA)] including D-3-hydroxybutyrate [hereinafter abbreviated as P (3HB)]. As typical examples other than P (3HB), 3HB and D-3-hydroxyvalerate (3H
V) copolymers [P. A. Holmes et al
(ICI), Eur. Pat. Appl. 005245
9 (1981)], a copolymer of 3HB and 4-hydroxybutyrate (4HB) [Y. Doi et al. ，
Macromolecules, 21, 2722 (19
88)]. Biopolyester accumulated in cells is known to exist as fine granules. The biopolyester content in the cells to be treated (hereinafter referred to as polymer content) is preferably high. Generally, the polymer content of dried bacterial cells is preferably 20% by weight or more. A polymer content of 50% by weight or more is particularly preferable in terms of efficiency of separation operation and purity of the separated polymer. The aqueous suspension refers to the culture suspension itself after completion of the culture, or a suspension of cells separated from the culture solution by centrifugation or the like in water. The suspension concentration of the bacterial cells is 150 g bacterial cells / 1 or less, preferably 100 g bacterial cells / 1 or less in terms of dry bacterial cells.

In the method of the present invention, the aqueous suspension is introduced into a pressure-resistant container having a minute opening and subjected to high pressure.
In this way, a large shearing force acts on the bacterial cells pushed out from the opening, and it is presumed that the bacterial cells are destroyed and the separation of biopolyester is promoted. A device composed of such a pressure-resistant container and a pressurizing mechanism is represented by a high-pressure homogenizer with a circulation device. Therefore, the biopolyester separation method of the present invention can be carried out by using a high-pressure homogenizer. The temperature setting of the high pressure homogenizer is 40
To 100 ° C, preferably 60 to 100 ° C. It is also preferable that the suspension is heated to a set temperature before the introduction of the high pressure homogenizer. The pressure applied to the suspension introduced into the high-pressure homogenizer depends on the device, but is 500-
Preference is given to working at 1500 kgf / cm ³ .

As a high pressure homogenizer with a circulation device,
Manton Gorlin (German APV / Gorlin), Minilab (Denmark APV Rani), Bran-Rube cell crusher (Bran + Luebbe, Germany), Microfluidizer (Microflulu, USA)
idics company) or the like can be used. It is well known that these devices are generally used for dispersion, emulsification, cell crushing and the like by applying pressure. Since heating in the high-pressure homogenizer is essential in the present invention, a similar type of high-pressure homogenizer, but a non-heating type French press, is not suitable for the present invention. It is known to use a French press to isolate biopolyesters in microorganisms on an experimental small scale (Helmut Bra
ndl et al, Advances in Bioc
chemical Engineering Biote
chology. 41, 77-93 (1990)),
No example is known of realizing the cooperative effect of separation by heating, which is a technical feature of the present invention.

By the above treatment operation, the bacterial cell wall can be efficiently destroyed in a short time, and the biopolyester can be separated from the bacterial cells in a granular form. When the bacterial cell wall is destroyed, a water-soluble polymer substance such as nucleic acid is eluted outside the cell, so that the concentration of the suspension once rises, but the shearing force causes the formation of a polymer such as nucleic acid. Perhaps because the cleavage also occurs, the viscosity of the suspension decreases again, and the biopolyester can be easily separated by the subsequent centrifugation operation, filtration operation, or the like. The cell concentration of the suspension before the treatment can be up to 150 g / l in terms of dry cells, so that it is not necessary to dilute the cell concentration after the usual culture. In the present invention, by heating the suspension, the bacterial cell wall becomes brittle and easily broken. Therefore, by applying a shearing force to the suspension, efficient treatment can be performed in a short time. In addition, the biopolyester granules can be taken out from the cells without breaking them.

[0011]

EXAMPLE The microorganism used in this example is a microorganism belonging to the genus Alcaligenes Alcaligenes liporitica (Al
caligenes lipolytica) AK2
01 (JP-A-5-64592), after culturing, P (3H
Centrifuge the bacteria containing about 50 wt% B) (8000r
pm, 10 min. Centrifuge KUBOTA 681
(0 use), the water was added to the paste-like microbial cells to obtain an aqueous suspension of 40 g microbial cells / l. Using the aqueous suspension, Examples 1 to 3 and Comparative Examples 1 and 2 shown below were performed.

P (3HB) obtained by the operations of Examples 1 to 3 and Comparative Examples 1 and 2 was subjected to gas chromatography to check the purity and gel permeation chromatography (GPC) to determine the molecular weight distribution. Analysis was carried out.
For gas chromatography, the precipitates obtained in Examples 1 to 3 and Comparative Examples 1 and 2 were dried (105 ° C, 2 ° C).
After 4 hours, methanol / sulfuric acid (85/15 wt
% / Wt%) was used for the methanolysis to analyze intracellular polyesters as monomeric methyl esters,
The polymer content was determined. This is [H. Brandl
et al, Int. J. Biol. Macromo
l. , 11, 49-55 (1989)]. For GPC, the polyester in the sample (about 100 mg) was extracted with 150 ml of hot chloroform, the solution was concentrated and hexane was added to reprecipitate, and the precipitate was filtered and vacuum dried (2 hr) to give a 10 mg / 10 ml chloroform solution. Was measured.

(Example 1) 500 ml of the suspension of P (3HB) -containing cells was prepared. Pre-suspend the suspension at 90 ° C for about 5
After heating for a minute, the mixture is placed in APV Gorin's Manton Gorin. In this device, the suspension is filled with a gauge pressure of about 100.
Pressurized to 0 kgf / cm ³ (at this time, the temperature inside the apparatus is controlled to the temperature of the heated suspension), and instantaneously (about 10 ⁻⁶ to 10 ⁻¹⁰ ).
^After passing through a gap of about 0.02 mm for ^-5 sec), it is discharged into the air and a fluid shear force is applied. This series of operations was repeated 10 times by automatically circulating the suspension.
The treated suspension is centrifuged (2700 rpm, 10 mi
n) to obtain a precipitate. (Example 2) The same operation as in Example 1 was carried out except that the suspension was preheated at 70 ° C for about 5 minutes, and the heating temperature in the manton-goulin was 70 ° C. (Example 3) In this example, the same operation as in Example 1 was carried out except that the suspension was preheated at 70 ° C for about 5 minutes to change the manton-goulin operation (10 times) to 20 times.

Comparative Example 1 In this example, the same operation as in Example 1 was carried out except that the suspension was not preheated. (Comparative Example 2) In this Example, 100 ml of the suspension was prepared, heated to 80 ° C. with stirring (100 rpm) in a closed container with nitrogen substitution, and stirring was continued for 1 hour. The treated suspension was centrifuged (2700 rpm, 10 min) to obtain a precipitate. Table 1 shows the separation conditions of Examples 1 to 3 and Comparative Examples 1 and 2.

[0015]

[Table 1] (Note) Heating temperature: Examples 1 to 3 and Comparative Example 1 show preheating temperature, and Comparative Example 2 shows treatment heating temperature. Table 2 shows the results obtained by gas chromatography and GPC in Examples and Comparative Examples.

[0016]

[Table 2]

[0017]

According to the present invention, a new separation method has been developed which overcomes the drawbacks of the conventional methods. That is, by using a high pressure homogenizer and applying a shearing force under relatively mild conditions in an aqueous medium without using an organic solvent,
The biopolyester could be separated from the microorganism containing the biopolyester.

Claims (2)

[Claims] 1. An aqueous suspension of biopolyester-containing microorganisms is introduced into a pressure-resistant container, heated and kept in the temperature range of 40 to 100 ° C., a high pressure is applied to the suspension, and a minute opening portion of the container is introduced. A method for separating a biopolyester from a biopolyester-containing microorganism, which comprises applying a fluid shearing force to the microbe by ejecting the suspension from the microbe to separate a granular biopolyester. 2. The method according to claim 1, wherein the aqueous suspension is heated in the range of 40 to 100 ° C. before being introduced into the pressure resistant container.